Compositions and methods for the treatment of immunodeficiency

ABSTRACT

The present invention relates to compositions and methods for the treatment of immunodeficiency (e.g., primary immunodeficiency disease). In particular, the invention provides human plasma immunoglobulin compositions containing select antibody titers specific for a plurality of respiratory pathogens, methods of identifying human donors and donor samples for use in the compositions, methods of manufacturing the compositions, and methods of utilizing the compositions (e.g., for prophylactic administration and/or therapeutic treatment (e.g., passive immunization (e.g., immune-prophylaxis))).

This application is a continuation of U.S. application Ser. No.15/978,692, filed May 14, 2018, now allowed, which is a continuation ofU.S. application Ser. No. 15/811,486, filed Nov. 13, 2017, now U.S. Pat.No. 9,969,793, which is a continuation of U.S. application Ser. No.14/592,727, filed Jan. 8, 2015, now U.S. Pat. No. 9,815,886, whichclaims the benefit of U.S. Provisional Application No. 62/069,589, filedOct. 28, 2014, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thetreatment of immunodeficiency (e.g., primary immunodeficiency disease).In particular, the invention provides human plasma immunoglobulincompositions containing select antibody titers specific for a pluralityof respiratory pathogens, methods of identifying human donors and donorsamples for use in the compositions, methods of manufacturing thecompositions, and methods of utilizing the compositions (e.g., forprophylactic administration and/or therapeutic treatment (e.g., passiveimmunization (e.g., immune-prophylaxis))).

BACKGROUND OF THE INVENTION

While most people have intact immune systems that serve to protect themfrom the wide variety of infectious organisms that commonly infectpeople including viruses, bacteria and fungi, many individuals haveimpaired or compromised immunity. There are many components to theimmune system all of which cooperate to reject foreign invadingpathogens. The humoral immune system that produces circulating antibodyis one of the principal components that is often found to be lacking inimmunocompromised individuals either at birth or may be a defect that isacquired. Immunodeficiency may be classified as primary or secondary.

Primary Immunodeficiency Diseases (PIDD) are a group of more than 150diseases in which part of a subject's immune system is missing or doesnot function normally. To be considered a primary immunodeficiency, thecause of the immune deficiency must not be secondary in nature (e.g.,caused by other disease, drug treatment, or environmental exposure totoxins). Most primary immunodeficiencies are genetic disorders and arediagnosed in children, although less severe forms may not be recognizeduntil adulthood. About 1 in 500 people are born with a primaryimmunodeficiency.

Most immunodeficiencies (e.g., primary and secondary) result in a faultyhumoral or cell mediated immune response toward infectious pathogens.The absence of a healthy, properly functioning humoral immune system(that part of the immune system required for generation of antibodiesthat are ultimately responsible for eradicating infection) renders aperson susceptible to many infections. Infusion of immunoglobulin hasbeen shown to reconstitute the ability of these immune defectiveindividuals to defend themselves against infection

Commercially available immunoglobulins are derived from pooled humanserum, collected, processed, and distributed for sale by the blood andplasma products industry. The first purified human immunoglobulin G(IgG) preparation used clinically was immune serum globulin which wasproduced in the 1940s (Cohn, E. J., et al “J. Am Chem. Soc., 68:459-475(1946)) and Oncely, J. L. et al., J. Am Chem Soc. 71:541-550 (1949). Theimmunoglobulin produced by this method demonstrated a moleculardistribution having a high molecular weight, when analyzed by way ofhigh resolution size exclusion chromatography. Immunoglobulin hashistorically been used primarily to prevent infections in patients whoare immune deficient. Immunoglobulin obtained from the plasma ofthousands of different donors contains antibodies to many of thepathogens that the donor individuals have encountered in their lifetimeand it is these antibodies when infused into patients with PIDD thatprevent them from suffering serious infections

However, significant limitations exist with currently availableimmunoglobulin products. Since immunoglobulin from thousands of randomdonors is pooled the antibody titers to the many infectious organisms(e.g., pathogens) for which protection is sought varies greatly and veryoften is not sufficient to meet the immune needs of the immunesuppressed individual (e.g., in case of a serious infection with apathogen).

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for thetreatment of immunodeficiency (e.g., primary immunodeficiency disease).In particular, the invention provides pooled human plasma immunoglobulincompositions, methods of identifying human plasma for use in thecompositions, methods of manufacturing the compositions, and methods ofutilizing the compositions (e.g., for prophylactic administration and/ortherapeutic treatment (e.g., passive immunization (e.g.,immune-prophylaxis))).

Accordingly, in one embodiment, the invention provides a compositioncomprising pooled plasma samples obtained from 1000 or more selectedhuman subjects (e.g., human plasma donors), wherein the pooled plasmacomprises elevated levels (e.g., selected, consistent and/orstandardized levels), compared to the pathogen-specific antibody titersfound in a mixture of plasma samples obtained from 1000 or more randomhuman subjects (e.g., human plasma donors), of pathogen-specificantibody titers to one or more (e.g., two, three, four, or more)respiratory pathogens. The invention is not limited by the type ofrespiratory pathogens for which the pooled plasma comprises elevatedlevels of pathogen-specific antibody titers. The pooled plasmacomposition may comprise elevated levels of pathogen-specific antibodytiters to one or more of respiratory syncytial virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus, coronavirus, S. pneumonia, H. influenza, L.pneumophila, group A Streptococcus, or any other respiratory pathogenknown in the art or described herein. In another embodiment, the pooledplasma from selected plasma donors comprises elevated levels, comparedto the pathogen-specific antibody titers found in a mixture of plasmasamples obtained from 1000 or more random human subjects, ofpathogen-specific antibody titers to two or more respiratory pathogensdescribed herein. In still another embodiment, the pooled plasmacomprises elevated levels, compared to the pathogen-specific antibodytiters found in a mixture of plasma samples obtained from 1000 or morerandom human subjects, of pathogen-specific antibody titers to three ormore respiratory pathogens described herein. In one embodiment, thepooled plasma comprises a respiratory syncytial virus-specific antibodytiter that is at least 2 fold greater (e.g. 2 fold, 3 fold, 4 fold, 5fold 6 fold, 7 fold, 8 fold, 9 fold, 10 fold or more) than therespiratory syncytial virus-specific antibody titer found in a mixtureof plasma samples obtained from 1000 or more random human subjects. Inanother embodiment, the pooled plasma comprises pathogen-specificantibody titers to at least two or more respiratory pathogens selectedfrom respiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirus,coronavirus, S. pneumonia, H. influenza, L. pneumophila, and group AStreptococcus that are each significantly elevated (e.g., at least 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 4.0, 5.0, 6.0, 7.0, 8.0 or more fold) compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects. In another embodiment,the pooled plasma comprises pathogen-specific antibody titers to atleast three or more respiratory pathogens selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus, coronavirus,S. pneumonia, H. influenza, L. pneumophila, and group A Streptococcusthat are each elevated at least 1.5 fold compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects. In still anotherembodiment, the pooled plasma comprises pathogen-specific antibodytiters to at least four or more respiratory pathogens selected fromrespiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirus,coronavirus, S. pneumonia, H. influenza, L. pneumophila, and group AStreptococcus that are each elevated at least 1.5 fold compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects. In one embodiment, thepooled plasma comprises plasma samples obtained from 1000-3000 or more(e.g., more than 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000 ormore) human subjects. In one preferred embodiment, the pooled plasmacomprises plasma samples obtained from 1000-1100 human subjects. In oneembodiment, the composition comprising pooled plasma samples furthercomprises a pharmaceutically acceptable carrier (e.g., natural and/ornon-naturally occurring carriers). In one embodiment, the pooled plasmacomposition is utilized to prepare immunoglobulin (e.g., for intravenousadministration to a subject). In one embodiment, the pooled plasmacomposition and/or immunoglobulin provides a therapeutic benefit to asubject administered the composition that is not achievable viaadministration of a mixture of plasma samples obtained from 1000 or morerandom human subjects and/or immunoglobulin prepared from same. Theinvention is not limited by the type of therapeutic benefit provided.Indeed, a variety of therapeutic benefits may be attained includingthose described herein. In one embodiment, the pooled plasma and/orimmunoglobulin possesses enhanced viral neutralization propertiescompared to a mixture of plasma samples obtained from 1000 or morerandom human subjects or immunoglobulin prepared from same. For example,in one embodiment, the pooled plasma possesses enhanced viralneutralization properties against one or more (e.g., two, three, four,five or more) respiratory pathogens (e.g., described herein). In afurther embodiment, the enhanced viral neutralization properties reduceand/or prevent infection in a subject administered the composition for aduration of time that is longer than, and not achievable in, a subjectadministered a mixture of plasma samples obtained from 1000 or morerandom human subjects. For example, in one embodiment, immunoglobulinprepared from pooled plasma according to the invention (e.g.,characterized, selected and blended according to the invention) that isadministered to a subject results in a significant, concentrationdependent anti-RSV neutralization activity and/or other respiratorypathogen (e.g., influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus, coronavirus,S. pneumonia, H. influenza, L. pneumophila, and group A Streptococcus)specific neutralization activity that is not achieved or achievableusing immunoglobulin prepared from randomly pooled plasma samples (e.g.,over a period of hours, days, weeks or longer). In one embodiment, thetherapeutic benefit of a pooled plasma and/or immunoglobulin of theinvention is enhanced viral neutralization properties that reduce orprevent infection in a subject administered the pooled plasma and/orimmunoglobulin for a duration of time that is longer than, and notachievable in, a subject administered a mixture of pooled plasma and/orimmunoglobulin prepared from same obtained from 1000 or more randomhuman subjects. In another embodiment, the therapeutic benefit of pooledplasma and/or immunoglobulin of the invention is a therapeutic and/orprotective level of antibody titers for measles, polio and/ordiphtheria. In one embodiment, the therapeutic benefit is a significantreduction in viral load in the lung and/or nose of an immunocompromisedsubject administered the pooled plasma and/or immunoglobulin compared toa control subject not receiving same. In a further embodiment, thepooled plasma and/or immunoglobulin significantly reduces lunghistopathology in an immunocompromised subject administered the pooledplasma and/or immunoglobulin compared to a control subject not receivingsame. In yet a further embodiment, the pooled plasma and/orimmunoglobulin significantly reduces the level of pathogenic viral RNAin a tissue selected from lung, liver and kidney in an immunocompromisedsubject administered the pooled plasma and/or immunoglobulin compared toa control subject. In one embodiment, a subject administeredimmunoglobulin prepared from pooled plasma according to the inventiondisplays a mean fold increase in anti-RSV neutralization titer that isat least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold or more at a time point of at least 1-14days (e.g., 14 day, 15 days, 16 days, 17 days, 18 days, 19 days or more)post administration of the immunoglobulin. The invention is not limitedby the amount of immunoglobulin administered to a subject. In oneembodiment, a subject is administered between 250-2500 mg/kg of theimmunoglobulin one time, or daily for two or more days (e.g., 2, 3, 4,or more consecutive days). In one embodiment, a subject is administered1500 mg/kg of immunoglobulin on day one and 750 mg/kg immunoglobulin onday 2. In another embodiment, a subject is administered 750 mg/kg ofimmunoglobulin on day one and 750 mg/kg immunoglobulin on day 2. In oneembodiment, the pooled plasma and/or immunoglobulin prepared from samereduces the incidence of infection in a subject administered thecomposition. In another embodiment, a pooled plasma and/orimmunoglobulin prepared from same reduces the number of days a subjectadministered the pooled plasma and/or immunoglobulin is required to beadministered antibiotics (e.g., to treat infection). In yet anotherembodiment, a pooled plasma and/or immunoglobulin prepared from sameincreases the trough level of circulating anti-respiratory pathogenspecific antibodies in a subject (e.g., increases the level ofneutralizing titers specific for respiratory pathogens (e.g., therebyproviding protective levels of anti-respiratory pathogen specificantibodies between scheduled dates of administration of the pooledplasma and/or immunoglobulin prepared from same that are not maintainedin a subject administered a mixture of plasma samples obtained from 1000or more random human subjects or immunoglobulin prepared from same)).

In another embodiment, the invention provides an immunotherapeuticcomposition comprising pooled plasma samples obtained from 1000 or moreselected human subjects, wherein the pooled plasma comprises elevatedlevels, compared to the pathogen-specific antibody titers found in amixture of plasma samples obtained from 1000 or more random humansubjects, of pathogen-specific antibody titers to two or morerespiratory pathogens selected from respiratory syncytial virus,influenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus, coronavirus, S. pneumonia,H. influenza, L. pneumophila, and group A Streptococcus; and apharmaceutically acceptable carrier. In one embodiment, animmunotherapeutic composition provided herein further comprises one ormore biologically active agents. The invention is not limited to thetype of biologically active agent/material. Indeed, a variety ofbiologically active agents/materials may be used including, but notlimited to, antibodies, anti-toxin material, anti-inflammatory agent,anti-cancer agent, antimicrobial agent, therapeutic agent,antihistamine, cytokine, chemokine, vitamin, mineral, or the like. Inone embodiment, the biologically active agent is an anti-toxin agent. Inone embodiment, the anti-toxin agent is a mono-specific, bi-specific ormulti-specific antibody with specificity toward a viral, bacterial orfungal toxin. In a further embodiment, the bacterial or fungal toxin isselected from Botulinum neurotoxin, Tetanus toxin, E. coli toxin,Clostridium difficile toxin, Vibrio RTX toxin, Staphylococcal toxins,Cyanobacteria toxin, and mycotoxins. In another embodiment, theimmunotherapeutic composition further comprises an aliquot of a singleor multiple monoclonal antibodies with a single or multiplespecificities (e.g., the immunogenic composition may be spiked with oneor more antibodies or biologically active material (e.g., a monoclonalantibody of any specificity, an anti-toxin agent, etc.)). The inventionis not limited by the type of one or more antibodies that are added to(e.g., spiked into) the immunogenic composition. Indeed, any one or moreantibodies (e.g., specific for a pathogen or pathogen product) may beused including, but not limited to standard antibodies, bi-specificantibodies, multi-specific antibodies, or the like known in the art(e.g., specific for one or a multiplicity of antigens).

In another embodiment, the invention provides a method of providingimmunotherapy to a subject (e.g., a subject in need thereof (e.g., asubject with disease or at risk for disease)) comprising administeringto the subject a therapeutically effective amount of a compositioncomprising pooled plasma samples obtained from 1000 or more selectedhuman subjects, wherein the pooled plasma comprises elevated levels,compared to the pathogen-specific antibody titers found in a mixture ofplasma samples obtained from 1000 or more random human subjects, ofpathogen-specific antibody titers to one or more (e.g., two, three,four, five or more) respiratory pathogens selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus, coronavirus,S. pneumonia, H. influenza, L. pneumophila, and group A Streptococcus.In one embodiment, the immunotherapy is used to prophylactically treatinfection associated with a microbial pathogen. In another embodiment,the immunotherapy is used to therapeutically treat infection associatedwith a microbial pathogen. In one embodiment, the subject has animmunodeficiency. The invention is not limited by the type ofimmunodeficiency. Indeed, the invention may be used to provideimmunotherapy compositions and methods to an immunocompromised patient,a subject at elevated risk of infection (e.g., experiencing an extendedhospital stay, and/or anticipating direct exposure to multiple specificpathogens), and/or an individual whose immune response has been rendereddeficient (e.g., from a disease of the immune system, from a diseasethat depresses immune functions (e.g., AIDS, idiopathic thrombocytopenicpurpura (ITP), etc.) from a therapy (e.g., chemotherapy) that results ina suppressed immune system). In one embodiment, immunotherapy methodsprovided herein arrest the progression of the immune deficiency andenable at least a partial recovery from the immune deficiency. Anadvantage of the compositions and methods described herein is that manyembodiments do not require the subject to be given additional drugs totreat their risk of infection, and therefore they are spared adverseside effects or interactions with other therapies. Another advantage ofthe compositions and methods described herein is that the compositionsand methods may be used to treat or prevent disease wherein drugs orother conventional treatments do not exist to treat or prevent thedisease (e.g., compositions and methods of the invention can be used totreat and/or prevent infection with respiratory syncytial virus). Yetanother advantage of the compositions and methods of the invention isthat the compositions and methods of the invention may be used to treatinfection when the underlying infectious agent is unknown. Moreover, inone embodiment, compositions and methods of the invention are utilizedfor prophylactic and/or therapeutic treatment of infection for whichthere exists no known cure (e.g., various viral illnesses). In someembodiments, compositions and methods of the invention are utilized forprophylactic and/or therapeutic treatment of infections (e.g., that areresistant to existing treatment (e.g., antibiotic resistantdisease/infection (e.g., vancomycin resistant staphylococci)). In someembodiments, compositions and methods of the invention are utilized forprophylactic and/or therapeutic treatment of a subject that harbors anon-competent immune system (e.g., in which treatment with antibiotic orother conventional antimicrobial therapy would have little to no value).In other embodiments, compositions and methods of the invention areutilized to reduce the risk of a subject developing a respiratoryinfection. The invention is not limited by the type of subject treatedwith the compositions and methods of the invention. Indeed, a variety ofsubjects may be so treated, including, but not limited to, a subject atrisk of developing an infection (e.g., respiratory or other type ofinfection (e.g., thereby reducing the risk of developing infection in asubject having an elevated risk of infection)). In one embodiment, asubject treated with a composition of the invention has or is diagnosedas having a primary immunodeficiency disease (PIDD). In anotherembodiment, the subject is an end stage renal disease (ESRD) patient;cancer patient on immunosuppressive therapy, AIDS patient, diabeticpatient, neonate, transplant patient, patient on immunosuppressiontherapy, patient with PIDD and other immune deficiencies, patient withmalfunctioning immune system, autoimmune disease patient, an elderlyperson in an extended care facility, patient with autoimmune disease onimmunosuppressive therapy, transplant patient, patient with invasivesurgical procedure, burn patient, or other patient in acute caresetting. In one embodiment, the immunotherapy provides the subject withprophylactic immunity against two or more pathogens selected fromClostridium botulinum, cytomegalovirus (CMV), Corynebacteriumdiphtheriae, hepatitis A virus, measles virus, hepatitis B virus,Hepatitis C virus, human immunodeficiency virus (HIV), rabies virus,tetanus, vaccinia virus, Pseudomonas aeruginosa, varicella-zoster virus,influenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus, coronavirus and respiratorysyncytial virus (RSV). However, the invention is not so limited. Indeed,immunotherapy with the compositions and methods of the invention mayprovide the subject with prophylactic immunity to any of the microbialpathogens described herein. In another embodiment, the immunotherapy isused to treat infection in the subject caused by Clostridium botulinum,cytomegalovirus (CMV), Corynebacterium diphtheriae, hepatitis A virus,measles virus, hepatitis B virus, Hepatitis C virus, humanimmunodeficiency virus (HIV), rabies virus, tetanus, vaccinia virus,Pseudomonas aeruginosa, varicella-zoster virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus, coronavirus and/or respiratory syncytial virus(RSV), or any microbial pathogen described herein.

In another embodiment, the invention provides a method of producing apooled plasma composition, comprising obtaining plasma samples fromhuman subjects; characterizing the pathogen-specific antibody titer,within a subset of the plasma samples, for one or more respiratorypathogens selected from respiratory syncytial virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus and coronavirus; selecting, based upon the antibodytiters characterized, plasma samples that have elevated levels, comparedto a control value (e.g., the pathogen-specific antibody titers found ina mixture of plasma samples obtained from 1000 or more random humansubjects), of pathogen-specific antibody titers to one or morerespiratory pathogens selected from respiratory syncytial virus,influenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus and coronavirus; pooling theselected plasma samples with other plasma samples to generate the pooledplasma composition, wherein the pooled plasma composition comprisespathogen-specific antibody titers to one or more respiratory pathogensselected from respiratory syncytial virus, influenza A virus, influenzaB virus, parainfluenza virus type 1, parainfluenza virus type 2,metapneumovirus and coronavirus, the one or more titers being elevatedat least 1.5 fold compared to a control value (e.g., thepathogen-specific antibody titers in a mixture of plasma samplesobtained from 1000 or more random human subjects). In one embodiment,the method comprises selecting, based upon the antibody titerscharacterized, plasma samples that have elevated levels, compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects, of pathogen-specificantibody titers to two or more respiratory pathogens selected fromrespiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirusand coronavirus. In a further embodiment, the method comprisesselecting, based upon the antibody titers characterized, plasma samplesthat have elevated levels, compared to the pathogen-specific antibodytiters found in a mixture of plasma samples obtained from 1000 or morerandom human subjects, of pathogen-specific antibody titers to three,four or more respiratory pathogens selected from respiratory syncytialvirus, influenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus and coronavirus. In oneembodiment, the pooled plasma composition comprises pathogen-specificantibody titers to at least two or more respiratory pathogens selectedfrom respiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirusand coronavirus that are each elevated at least 1.5 fold compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects. In one embodiment, thepooled plasma composition comprises pathogen-specific antibody titers toat least three or more respiratory pathogens selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus andcoronavirus that are each elevated at least 1.5 fold compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects. In another embodiment,the pooled plasma composition comprises a respiratory syncytialvirus-specific antibody titer that is at least 2 fold greater (e.g., 2,2.5, 3, 3.5, 4.5, 5, 6, 7, 8, 9, 10 fold or more) than the respiratorysyncytial virus-specific antibody titer found in a mixture of plasmasamples obtained from 1000 or more random human subjects. For example,in one embodiment, the invention provides a method of producing a pooledplasma composition containing a specific, elevated antibody titer forrespiratory syncytial virus (RSV) and a specific, elevated antibodytiter for one or more other respiratory pathogens, from at least 1000human plasma donors, comprising obtaining plasma samples from selectedhuman plasma donors and non-selected human plasma donors, wherein theselected human plasma donors comprise high titer selected human donorsand non-high titer selected human donors, wherein the selected humandonors are identified via characterizing the specific titer ofantibodies to respiratory syncytial virus in a plasma sample from ahuman donor, wherein characterizing the specific titer of antibodies torespiratory syncytial virus comprises a first, plasma screening assayutilized to assess neutralizing activity in the plasma sample; and asecond screening assay characterizing the specific antibody titer of thepurified immunoglobulin fraction of each plasma sample identified asdisplaying the top 20% of neutralizing activity using the first, plasmascreening assay, wherein a purified immunoglobulin fraction possessingan RSV neutralization titer of 1800 or above is used to identify aplasma sample containing an elevated antibody titer for one or morerespiratory pathogens selected from parainfluenza virus 1, parainfluenzavirus 2, coronavirus OC43, coronavirus 229E, influenza A virus,influenza B virus, and metapneumovirus, and used to categorize a humandonor as a high titer selected human donor; and pooling 1000 or moreplasma samples from high titer selected human donors, non-high titerselected human donors, and non-selected human donors in order togenerate the pooled plasma composition, wherein 10-65%, 20-55%, 30-50%,40-50% (e.g., in a preferred embodiment, less than 50% (e.g., about30-45%, 35-45%, 40-45%)) of the 1000 or more plasma samples are fromhigh titer selected human donors; and wherein the pooled plasmacomposition comprises an RSV specific antibody titer that is at least 3times greater than the RSV specific antibody titer in a control sample,and an antibody titer for one or more respiratory pathogens selectedfrom parainfluenza virus 1, parainfluenza virus 2, coronavirus OC43 andcoronavirus 229E that is at least about 1.5 times greater (e.g., about1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 times or more)than the antibody titer in the control sample, wherein the controlsample is a mixture of plasma samples obtained from 1000 or more randomhuman subjects. Pooled plasma compositions produced according to themethods described herein are also provided. In one embodiment, less than50% of the total volume of the pooled plasma composition (e.g., inpreferred embodiments, about 30-45%, 35-45%, 35-40%)) comprises plasmaobtained from high titer selected human donors; about 55-70% (e.g., inpreferred embodiment, about 55-75%, 55-65%) of the total volume of thepooled plasma composition comprises plasma obtained from non-high titerselected human donors, and about 3-20% of the total volume of the pooledplasma composition comprises plasma from non-selected human donors. Inone embodiment, the pooled plasma composition provides a therapeuticbenefit to a subject administered the composition that is not achievablevia administration of a mixture of plasma samples obtained from 1000 ormore random human subjects. The invention is not limited by the type oftherapeutic benefit provided. Indeed, a variety of therapeutic benefitsmay be attained including those described herein. In one embodiment, thepooled plasma composition possesses enhanced viral neutralizationproperties compared to a mixture of plasma samples obtained from 1000 ormore random human subjects. In a further embodiment, the enhanced viralneutralization properties reduce and/or prevent infection in a subjectadministered the composition for a duration of time that is longer than,and not achievable in, a subject administered a mixture of plasmasamples obtained from 1000 or more random human subjects.

In one embodiment, the invention provides the use of neutralizingantibody titer to RSV (or other respiratory pathogen) as a biomarker toidentify plasma donors that are high/strong responders in general toother respiratory pathogens (e.g., influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirus,coronavirus, S. pneumonia, H. influenza, L. pneumophila, and group AStreptococcus). In a further embodiment, the invention provides a methodof using the level of a respiratory pathogen specific neutralizingantibody titer (e.g., RSV neutralizing antibody titer) detected in aplasma donor sample as a biomarker that indicates and/or providesinformation regarding the level of respiratory pathogen specificneutralizing antibody titers within the sample to one or more ofinfluenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus and coronavirus. The use ofsuch a biomarker makes possible the ability to identify donors andplasma (e.g., high titer selected donor and/or donor sample) that can beblended with non-high titer selected donor plasma or non-selected donorplasma to provide a pooled plasma composition of the invention. In oneembodiment, a plasma sample identified as having a specific antibodytiter to respiratory syncytial virus (e.g., via a first, plasmascreening assay utilized to assess neutralizing activity in the plasmasample; and a second screening assay characterizing the specificantibody titer of the purified immunoglobulin fraction of each plasmasample identified as displaying the top 20% of neutralizing activityusing the first, plasma screening assay) of about 1800 or above (e.g.,about 1800, 1850, 1900, 1950, 2000, 2050, 2100 or more) is used toidentify a plasma sample containing an elevated antibody titer (e.g.,wherein the elevated titer is a titer that is at least about 1.5 timesgreater (e.g., about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0times or more) than the antibody titer in a control sample, wherein thecontrol sample is a mixture of plasma samples obtained from 1000 or morerandom human subjects) for one or more respiratory pathogens selectedfrom parainfluenza virus 1, parainfluenza virus 2, coronavirus OC43,coronavirus 229E, influenza A virus, influenza B virus, andmetapneumovirus.

In another embodiment, the invention provides hyperimmune globulincompositions (e.g., adenoviral hyperimmune globulin compositions, RSVhyperimmune globulin compositions pneumococcal hyperimmune globulincompositions, etc.) and methods of generating and using the same (e.g.,for passive immunization of patients susceptible to, or suffering from,infection (e.g., bacterial infection (e.g., associated withStreptococcus pneumonia, Haemophilus influenza, etc.), viral infection(e.g., associated with adenovirus, herpes virus, influenza virus,rhinovirus, etc.), fungal infection (e.g., associated with Aspergillusfumigatus, Cladosporium or other fungi) and/or exposure to microbialtoxins and/or harmful agents (e.g., animal venom))). In one embodiment,pooled human plasma samples (e.g., with elevated titer of desiredantibodies (e.g., targeted to specific pathogens and/or that areidentified as containing a specific desired property (e.g., enhancedanti-inflammatory property))) are combined to produce tailoredimmunoglobulin pools (e.g., that possess a desired characteristic thatis not achievable in the absence of combining the pools (e.g., greaterlevel of protection against infection, inflammation or toxins)) withelevated titer of antibodies against multiple specific pathogens orpathogen products (e.g., toxins).

In one embodiment, the invention provides compositions and methods forobtaining a composition comprising pooled plasma samples (e.g., plasmafrom a plurality of donors (e.g., that contain elevatedmicrobial-specific antibody titers (e.g., high titer of viral-,bacterial-, and/or fungal-pathogen specific immunoglobulin))). Asdescribed herein, the plasma pool may be utilized as or in a therapeuticcomposition (e.g., to treat and/or prevent viral, bacterial and/orfungal infection). In one embodiment, the invention provides acomposition comprising pooled plasma samples (e.g., a therapeuticcomposition) comprising plasma from a plurality of donors (e.g., 1000 ormore human donors), wherein all or a subset of the plurality of donorspossess a high titer of microbial pathogen-specific antibodies to one ora plurality of microbial pathogens as a result of administration of oneor a plurality of immunogenic compositions comprising microbial antigensto the plurality of donors. The invention is not limited by the methodof obtaining an antibody pool. Indeed, a variety of methods may beutilized, including, but not limited to, administering one or moremicrobial (e.g., viral, bacterial and/or fungal) antigens to a hostsubject to generate enhanced expression of microbial (e.g., viral,bacterial and/or fungal) specific antibodies and obtaining serum orplasma containing microbial (e.g., viral, bacterial and/or fungal)specific, enhanced high titer antibody pools from the donor or pluralityof donors. In some embodiments, the plasma/serum is purified and/orconcentrated (e.g., in order to concentrate microbial (e.g., viral,bacterial and/or fungal) specific immunoglobulin present therein (e.g.,prior to providing (e.g., administration) to a subject). The inventionis not limited by the one or more microbial (e.g., viral, bacterialand/or fungal) antigens utilized to generate enhanced expression ofmicrobial (e.g., viral, bacterial and/or fungal) specific antibodies.Indeed, a variety of microbial (e.g., viral, bacterial and/or fungal)antigens may be utilized, including, but not limited to, conjugated andunconjugated microbial antigenic proteins or peptides, sugars(polysaccharides), cell wall components, viral antigens etc. In someembodiments, the viral, bacterial and/or fungal antigens utilized are,or are in the form of, a commercially available vaccine. Commerciallyavailable vaccines are well known to those in the field. By way ofexample, non-limiting examples of commercially available vaccines thatfind use in the invention include, but are not limited to, AdenovirusType 4 and Type 7 vaccine, Anthrax vaccine, BCG vaccine, Diphtheria andTetanus Toxoids, Diphtheria and Tetanus Toxoids, Diphtheria and TetanusToxoids and Acellular Pertussis vaccine, Diphtheria and Tetanus Toxoidsand Acellular Pertussis vaccine, Diphtheria and Tetanus Toxoids andAcellular Pertussis vaccine, Diphtheria and Tetanus Toxoids andAcellular Pertussis vaccine, Hepatitis B (recombinant) and InactivatedPoliovirus vaccine, Diphtheria and Tetanus Toxoids and AcellularPertussis, Inactivated Poliovirus and Haemophilus b Conjugate (TetanusToxoid Conjugate) vaccine, Haemophilus b Conjugate vaccine (e.g.,Meningococcal Protein Conjugate, Tetanus Toxoid Conjugate), recombinantHepatitis B vaccine, Hepatitis A vaccine, Hepatitis A Inactivated andHepatitis B (Recombinant (e.g., RECOMBIVAX HB, ENGERIX-B) vaccine, HumanPapillomavirus vaccine (e.g., multivalent, bivalent, quadrivalent (Types6, 11, 16, 18) vaccine), Influenza Virus vaccine (e.g., influenza A(H1N1) vaccine, monovalent vaccine, trivalent (e.g., Types A and B)vaccine, H5N1 vaccine, FLUMIST, FLUARIX, FLUVIRIN, AGRIFLU, FLUZONE,FLUXELVAX, FLUMIST quadrivalent,), Japanese Encephalitis Virus vaccine,Measles Virus vaccine, Measles and Mumps Virus vaccine, Measles, Mumps,and Rubella Virus vaccine, Measles, Mumps, Rubella and Varicella Virusvaccine, Meningococcal vaccine (e.g., Groups A, C, Y, and W-135Oligosaccharide Diphtheria CRM197 Conjugate vaccine), MeningococcalGroups C and Y and Haemophilus b Tetanus Toxoid Conjugate vaccine,Meningococcal Polysaccharide (Serogroups A, C, Y and W-135) DiphtheriaToxoid Conjugate vaccine, Meningococcal Polysaccharide vaccine (e.g.,Groups A, C, Y and W-135 Combined vaccine), Mumps Virus vaccine, Plaguevaccine, Pneumococcal vaccine (e.g., PNEUMOVAX23, Pneumococcal 7-valentConjugate vaccine, PREVNAR, Pneumococcal 13-valent Conjugate vaccine),Poliovirus vaccine, Rabies vaccine, Rotavirus vaccine, Rubella Virusvaccine, Smallpox (Vaccinia) vaccine, Tetanus and Diphtheria Toxoids(e.g., DECAVAC, TENIVAC), Tetanus Toxoid, Reduced Diphtheria Toxoid andAcellular Pertussis vaccine, Typhoid vaccine, Typhoid Vi Polysaccharidevaccine, Varicella Virus vaccine, Yellow Fever vaccine, and/or Zostervaccine. In some embodiments, the microbial antigen is a viral antigen(e.g., respiratory syncytial virus antigen), bacterial antigen (e.g., S.pneumoniae antigen) and/or a fungal antigen. In some embodiments, a S.pneumoniae antigen is a S. pneumoniae cell membrane sugar (e.g., apolysaccharide). In some embodiments, a S. pneumoniae antigen is aconjugate vaccine (e.g., conjugated to a carrier and/or adjuvant (e.g.,a protein or other carrier molecule). In some embodiments, a S.pneumoniae antigen is an unconjugated vaccine. In some embodiments, theconjugate vaccine or unconjugated vaccine contains 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or moredifferent antigens (e.g., from an equal number of different serotypes ofS. pneumonia). In some embodiments, the one or more different serotypesof S. pneumoniae include, but are not limited to, serotypes 1, 2, 3, 4,5, 6A, 6B, 7A, 7B, 7C, 7D, 7E, 7F, 8, 9A-9V, 12, 14, 18C, 19A-19F,23A-23F, and 25. In some embodiments, the one or more differentserotypes of S. pneumoniae are selected from any one of the more the 90different S. pneumoniae serotypes identified. In some embodiments, theone or more different serotypes of S. pneumoniae is newly identified.

In one embodiment, compositions are provided that comprise a pluralityof different types of antibodies (e.g., directed to different pathogens(e.g., viral pathogens, bacterial pathogens, eukaryotic pathogens,etc.), recognize different antigens, recognize different epitopes, etc.)and are enriched (e.g., elevated titer) for at least two differentantibodies or sets of antibodies (e.g., directed to different pathogens,recognize different antigens, recognize different epitopes, etc.). Inparticular embodiments, compositions comprise tailored antibody pools.In some embodiments, at least from about 0.01% to about 70% of the totalimmunoglobulin present in the composition is directed to one or moretargeted pathogens, although the invention is no so limited (e.g., thecomposition may comprise less than 0.01% or more than 70% ofimmunoglobulin directed to targeted pathogens). Immunoglobulin directedto targeted pathogens maycomprise >0.1% >2%, >5%, >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%,or >90% of the total immunoglobulin present in the composition. Incertain embodiments, a composition comprises two or more immunoglobulinsto targeted pathogens, each present at greater than 1% of totalimmunoglobulin present in the composition (e.g., two or moreimmunoglobulins to targeted pathogens present at greater than 1.5%,2.0%, 3.0%, 4.0%, 5.0% or more of total immunoglobulin, two or moreimmunoglobulins to targeted pathogens present at greater than 10% oftotal immunoglobulin, two or more immunoglobulins to targeted pathogenspresent at greater than 15% of total immunoglobulin, two or moreimmunoglobulins to targeted pathogens present at greater than 20% oftotal immunoglobulin, two or more immunoglobulins to targeted pathogenspresent at greater than 25% of total immunoglobulin, etc.).

Any suitable method for obtaining plasma, antibody samples, pooledplasma compositions and/or immunoglobulin from same are within the scopeof the present invention. Further, any suitable method for producing,manufacturing, purifying, fractionating, enriching, etc. antibodysamples and/or plasma pools is within the bounds of the presentinvention. Exemplary techniques and procedures for collecting antibodysamples and producing plasma pools are provide, for example, in: U.S.Pat. Nos. 4,174,388; 4,346,073; 4,482,483; 4,587,121; 4,617,379;4,659,563; 4,665,159; 4,717,564; 4,717,766; 4,801,450; 4,863,730;5,505,945; 5,582,827; 6,692,739; 6,962,700; 6,984,492; 7,045,131;7,488,486; 7,597,891; 6,372,216; U.S. Patent App. No. 2003/0118591; U.S.Patent App. No. 2003/0133929 U.S. Patent App. No. 2005/0053605; U.S.Patent App. No. 2005/0287146; U.S. Patent App. No. 2006/0110407; U.S.Patent App. No. 2006/0198848; U.S. Patent App. No. 2006/0222651; U.S.Patent App. No. 2007/0037170; U.S. Patent App. No. 2007/0249550; U.S.Patent App. No. 2009/0232798; U.S. Patent App. No. 2009/0269359; U.S.Patent App. No. 2010/0040601; U.S. Patent App. No. 2011/0059085; andU.S. Patent App. No. 2012/0121578; herein incorporated by reference intheir entireties. Embodiments of the present invention may utilize anysuitable combination of techniques, methods, or compositions from theabove listed references.

In some embodiments, plasma and/or antibody samples are obtained fromdonor subjects in the form of donated or purchased biological material(e.g., blood or plasma). In some embodiments, plasma and/or antibodysamples (e.g., blood, plasma, isolated antibodies, etc.) are obtainedfrom a commercial source. In some embodiments, a plasma and/or antibodysample, blood donation, or plasma donation is screened for pathogens,and either cleaned or discarded if particular pathogens are present. Inone embodiments, screening occurs prior to pooling a donor sample withother donor samples. In other embodiments, screening occurs afterpooling of samples. Antibodies, blood, and/or plasma may be obtainedfrom any suitable subjects. In some embodiments, antibodies, blood,and/or plasma are obtained from a subject who has recently (e.g., within1 year, within 6 months, within 2 months, within 1 month, within 2weeks, within 1 week, within 3 days, within 2 days, within 1 day) beenvaccinated against or been exposed to one or more specific pathogens. Incertain embodiments, a subject positive for antibodies to the pathogenof interest is administered antigens to that pathogen to increase titerof the desired antibodies. In some embodiments, a subject has producedantibodies and/or has elevated titer of antibodies against one or morespecific pathogens. In certain embodiments, a subject, whether negativeor positive for antibodies to a specific microbial pathogen isadministered one or more different viral, bacterial and/or fungalantigens/vaccines in order to increase titer of specific, desiredantibodies (e.g., viral-, bacterial- and/or fungal-specific antibodies).Pathogens to which a donor may have elevated titer of antibodiesinclude, but are not limited to: Clostridium botulinum, cytomegalovirus(CMV), Corynebacterium diphtheriae, hepatitis A virus, measles virus,hepatitis B virus, Hepatitis C virus, human immunodeficiency virus(HIV), rabies virus, tetanus virus, vaccinia virus, Pseudomonasaeruginosa, varicella-zoster virus, and respiratory syncytial virus(RSV), human immunodeficiency virus, hepatitis C virus, human papillomavirus, hepatitis B virus, or other human viral or bacterial pathogens.

In some embodiments, plasma samples known, identified, and/or selected(e.g., according to methods described herein) to contain elevated titerof a particular antibody (e.g., antibodies directed to RSV) or a set ofplasma samples are combined (e.g., pooled) to produce a compositioncomprising pooled plasma samples (e.g., with elevated titer ofantibodies directed to a particular pathogen or to a set of pathogens(e.g., RSV, and one or more other respiratory pathogens)). For example,a composition comprising pooled plasma samples is produced by poolingplasma samples obtained from selected human subjects and non-selectedhuman subjects, wherein the pooled plasma comprises elevated levels(e.g., elevated by about 20%, 30%, 40%, 50%, 60%, 70%, 85%, 90%, 100%,125%, 150%, 160%, 170%, 175%, 180%, 200%, 225%, 250%, 275%, 300%, 350%,400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%,1000% or more), compared to a control value (e.g., the pathogen-specificantibody titers found in a mixture of plasma samples obtained from 1000or more random human subjects), of pathogen-specific (e.g., RSVspecific, influenza A virus specific, influenza B virus specific,parainfluenza virus type 1 specific, parainfluenza virus type 2specific, metapneumovirus specific and/or coronavirus specific) antibodytiters. In a further embodiment, immune globulin is prepared from thepooled plasma (e.g., according to techniques and methods describedherein). In some embodiments, a composition comprising pooled plasmasamples is produced by pooling plasma samples obtained from selectedhuman donors and non-selected human donors, wherein the pooled plasmacomprises elevated levels, compared to the pathogen-specific antibodytiters found in a mixture of plasma samples obtained from 1000 or morerandom human subjects, of RSV-specific antibody titers (e.g.,individuals recently exposed to RSV, individuals recently vaccinated forRSV, etc) and other respiratory pathogen specific titers. In oneembodiment, a composition comprising pooled plasma samples and/or immuneglobulin prepared therefrom of the invention is a sterile solution witha pH of about 6.0-7.8 (e.g., 5.0-6.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6. 7.7, 7.8, or higher).In another embodiment, a composition comprising pooled plasma samplesand/or immune globulin prepared therefrom of the invention is preparedaccording US FDA standards for immune globulin preparation (e.g., 37 CFR§§ 640.100; 640.101; 640.102; 640.103; 640.104, Apr. 1, 2013). In oneembodiment, a composition comprising pooled plasma samples and/or immuneglobulin prepared therefrom of the invention (e.g., RSV-IVIG describedherein, in particular, in the Examples) possesses at least the minimumlevel of antibody titers to Corynebacterium diphtheria, measles virus,and polio virus recommended by the FDA (e.g., see 37 CFR § 640.104).

In one embodiment, a composition comprising pooled plasma samples and/orimmune globulin prepared therefrom of the invention comprises elevatedantibody titer levels, compared to a control antibody titer value (e.g.,the pathogen-specific antibody titer found in a mixture of plasmasamples obtained from 1000 or more random human subjects), ofpathogen-specific antibodies to respiratory syncytial virus and one ormore respiratory pathogens selected from, influenza A virus, influenza Bvirus, parainfluenza virus type 1, parainfluenza virus type 2,metapneumovirus and coronavirus, wherein the elevated levels of RSVspecific, influenza A virus specific, influenza B virus specific,parainfluenza virus type 1 specific, parainfluenza virus type 2specific, metapneumovirus specific and/or coronavirus specificantibodies are elevated at least 20%, 30%, 40%, 50%, 60%, 70%, 85%, 90%,100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, 450%,500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% ormore), compared to a control value (e.g., the pathogen-specific antibodytiter level found in a mixture of plasma samples obtained from 1000 ormore random human subjects). The invention provides a method, in oneembodiment, of generating the above described composition comprisingobtaining plasma samples from selected human donors and non-selectedhuman donors; pooling 1000 or more plasma samples from both selecteddonors and non-selected donors to generate the pooled plasmacomposition. In one embodiment, the plasma samples from selected humandonors and non-selected human donors are screened in order to confirmthe absence of bloodborne pathogens (e.g., before or after pooling). Ina further embodiment, selected human donors are identified viaidentifying the specific titer of antibodies to one or more respiratorypathogens selected from respiratory syncytial virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus and coronavirus. In a preferred embodiment, selectedhuman donors are identified via identifying the specific titer ofantibodies to respiratory syncytial virus. In a further embodiment, theselected human donors comprise high titer donors and medium titerdonors, wherein high titer donors comprise a pathogen specific antibodytiter that is 2-5 times, 5-8 times, 8-10 times, 10-14 times, 14 times orgreater than a standard value (the titer of pathogen specific antibodiespresent in a pool of plasma samples from 1000 or more random humansubjects), and wherein medium titer donors comprise a pathogen specificantibody titer that is the titer of pathogen specific antibodies presentin a pool of plasma samples from 1000 or more random human subjects orthat is only marginally higher (e.g., 5-20% higher) or marginally lower(e.g., 5-20% lower) than this value. In still a further embodiment, theselected human donors comprise high titer donors, medium titer donorsand low titers donors, wherein high titer donors comprise a pathogenspecific antibody titer that is 2-5 times, 5-8 times, 8-10 times, 10-14times, 14 times or greater than a standard value (the titer of pathogenspecific antibodies present in a pool of plasma samples from 1000 ormore random human subjects), wherein medium titer donors comprise apathogen specific antibody titer that is the titer of pathogen specificantibodies present in a pool of plasma samples from 1000 or more randomhuman subjects or that is only marginally higher (e.g., 5-20% higher) ormarginally lower (e.g., 5-20% lower) than this value, and wherein lowtiter donors comprise a pathogen specific antibody titer that is around20-50 percent the titer of pathogen specific antibodies present in apool of plasma samples from 1000 or more random human subjects.

In one embodiment, identifying antibody titer comprises a first, plasmascreening assay assessing neutralizing activity in a plasma sample, anda second screening assay assessing antibody titer in a purifiedimmunoglobulin fraction of the plasma sample. In one embodiment,neutralizing activity in plasma is measured via the absence of infectionby RSV of hepatocytes. In a further embodiment, the first plasmascreening assay assessing neutralization activity categorizes plasmasamples as high titer, medium titer, or low titer for RSV specificantibodies, wherein a high titer RSV donor/donor sample is one having anRSV neutralizing titer of about 7800 or greater, a medium titer RSVdonor/donor sample is one having an anti-RSV titer of about 3300-7799,and a low titer RSV donor/donor sample is one having an anti-RSV titerof about 1800-3299 (e.g., titer being calculated and assigned to adonor/donor sample as the dilution that give 50% inhibition of virusgrowth (that point which is 50% of the two extremes (saline plus virusis 100 growth and no virus added is 0 growth) according to methodsdescribed herein (See, e.g., Examples 1 and 2). In still a furtherembodiment, only plasma samples identified as displaying the top 20%-30%of neutralizing activity of all donors are processed to produce purifiedimmunoglobulin and subsequently screened using the second screeningassay. In a preferred embodiment, only plasma samples identified asdisplaying the top 20% of neutralizing activity of all donors areprocessed to produce purified immunoglobulin and subsequently screenedusing the second screening assay. In one embodiment, the secondscreening assay characterizes the RSV specific antibody titer of apurified immunoglobulin fraction of the plasma sample. In a preferredembodiment, a donor/donor sample that has an RSV neutralization titer ofat least 1800 in a purified immunoglobulin fraction of the plasma sampleis scored as a high titer donor. In a preferred embodiment, an RSVneutralization titer of at least 1800 is used to identify a donor/donorsample (e.g., plasma sample) comprising elevated levels of one or morerespiratory pathogens selected from influenza A virus, influenza Bvirus, parainfluenza virus type 1, parainfluenza virus type 2,metapneumovirus and coronavirus. In another embodiment, the pooledplasma composition comprises an RSV neutralization antibody titer of1800 or more. In one embodiment, less than half (e.g., about 10-20%,20-30%, 30-40%, 40-50%) of the 1000 or more donors are high titerdonors. In a preferred embodiment, about 30-45%, 35-45%, or 35-40% ofthe 1000 or more donors are high titer donors. In another embodiment,the invention provides a pooled plasma composition and/or immuneglobulin obtained from same prepared according to the above describedmethods. In one embodiment, the pooled plasma composition comprise about1800-2500 liters (e.g., about 1800, about 1900, about 2000, about 2100,about 2200, about 2300, about 2400 or about 2500 liters) of plasma from1000 donors with an RSV neutralization antibody titer of 1800 or more.In one embodiment, a pooled plasma composition of the inventioncomprises about 2200 liters of plasma from 1000 donors with an RSVneutralization antibody titer of 1800 or more, wherein less than 50% ofthe total volume of the pooled plasma composition (e.g., in preferredembodiments, about 30-45%, 35-45%, 35-40%)) comprises plasma obtainedfrom high titer selected human donors; about 55-70% (e.g., in preferredembodiment, about 55-75%, 55-65%) of the total volume of the pooledplasma composition comprises plasma obtained from non-high titerselected human donors, and about 3-20% of the total volume of the pooledplasma composition comprises plasma from non-selected human donors. In afurther embodiment, the pooled plasma composition comprisespathogen-specific antibody titers to at least two or more respiratorypathogens selected from influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirusand coronavirus that are each elevated at least about 1.5 fold (e.g.,about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 fold or more)compared to a control valued (e.g., the pathogen-specific antibodytiters found in a mixture of plasma samples obtained from 1000 or morerandom human subjects, or the pathogen-specific titers found in aconventional hyperimmune immune globulin (e.g., hyperimmune immuneglobulin for rabies (HYPERRAB, Grifols, Clayton, N.C.), hyperimmuneglobulin for hepatitis (e.g., HYPERHEP B, Talecris Biotherapeutics,Research Triangle Park, N.C.), hyperimmune globulin for RSV (e.g.,RESPIGAM, MEDIMMUNE, Inc.)). In one embodiment, the pooled plasmacomposition comprises at least the minimum titer of antibodies toCorynebacterium diphtheria, measles virus, and polio virus recommendedby the FDA (e.g., see 37 CFR § 640.104). In one embodiment, the pooledplasma composition comprises a respiratory syncytial virus-specificantibody titer that is at least 3 fold greater (e.g., 3, 4, 5, 6, 7, 8,9, 10 fold or more) than the respiratory syncytial virus-specificantibody titer found in a mixture of plasma samples obtained from 1000or more random human subjects. In one embodiment, the pooled plasmacomposition provides a therapeutic benefit to a subject administered thecomposition that is not achievable via administration of a mixture ofplasma samples obtained from 1000 or more random human subjects.Multiple types of therapeutic benefits are provided including, but notlimited to, inhibition of infection caused by RSV, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus and/or coronavirus in a subject administered thecomposition for a duration of time that is longer than and notachievable in a subject administered a mixture of plasma samplesobtained from 1000 or more random human subjects; significant reductionin viral load in the lung and/or nose (e.g., in an immunocompromisedsubject administered the composition compared to a control subject notreceiving the composition); significant reduction in lung histopathology(e.g., in an immunocompromised subject administered the compositioncompared to a control subject not receiving the composition); and/orsignificant reduction in the level of pathogenic viral RNA in lung,liver, kidney and/or other tissue (e.g., in an immunocompromised subjectadministered the composition compared to a control subject not receivingthe composition). In one embodiment, the pooled plasma composition lacksdetectable levels (e.g., detected using any method known in the art(e.g., recommended by the U.S. Food and Drug Administration)) of humanimmunodeficiency virus (HIV) 1 (HIV-1), HIV-2, Treponema pallidum,Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodiumvivax, Plasmodium knowlesi, hepatitis B virus(HBV), hepatitis C virus(HCV), prions, West Nile virus, parvovirus, Typanosoma cruzi, SARScoronavirus, and/or vaccinia virus. In one embodiment, each individualplasma sample used in a process or composition of the invention iscollected only at an FDA approved blood establishments and is tested byserological tests (e.g., FDA approved serological tests) for humanimmunodeficiency virus (HIV) 1 (HIV-1), HIV-2, Treponema pallidum,Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodiumvivax, Plasmodium knowlesi, hepatitis B virus(HBV), hepatitis C virus(HCV), prions, West Nile virus, parvovirus, Typanosoma cruzi, SARScoronavirus, and/or vaccinia virus. In another embodiment, an individualplasma sample and/or a pooled plasma composition of the invention istested for the presence of HIV-1, HIV-2, HBV, HCV, or other infectiousagent (e.g., pathogen) using Nucleic Acid Testing (NAT) and used in aprocess or composition of the invention only when the absence of thepathogens is confirmed.

The invention is not limited by the type of subject (e.g., mammal,non-human primate, human, etc.) administered or treated with acomposition of the invention (e.g., pooled plasma samples and/orimmunotherapeutic composition comprising same). Indeed, the subject maybe any subject in need of treatment with a composition of the invention(e.g., a subject infected with or susceptible to infection (e.g., due toan immune deficiency) with an infectious agent (e.g., any one or moreinfectious agents described herein (e.g., respiratory pathogens))). Insome embodiments, the subject is at elevated risk for infection (e.g.,by one or multiple specific pathogens (e.g., respiratory pathogens)).The subject may be a neonate. In some embodiments, the subject has animmunodeficiency (e.g., a subject receiving immunosuppressing drugs(e.g., a transplant patient), suffering from a disease of the immunesystem, suffering from a disease that depresses immune functions,undergoing a therapy (e.g., chemotherapy) that results in a suppressedimmune system, experiencing an extended hospital stay, and/or a subjectanticipating direct exposure to a pathogen or pathogens. For example, incertain embodiments, an immunocompromised subject is an end stage renaldisease (ESRD) patient; cancer patient on immunosuppressive therapy(e.g., chemotherapy, radiation), AIDS patient, diabetic patient,neonate, transplant patient (e.g., HSCT, BMT, Cord Blood,Haploidentical, and/or solid organ transplant patient), patient onimmunosuppression therapy (e.g., medical immunosuppression, steroids),patient with PIDD and other immune deficiencies, patient withmalfunctioning immune system, autoimmune disease patient, elderly personin an extended care facility, patient with autoimmune disease onimmunosuppressive therapy, transplant patient, patient with invasivesurgical procedure, burn patient, or other patient in an acute caresetting. In some embodiments, the subject treated with the compositionsand/or methods of the invention include subjects with a healthy ornormal immune system (e.g., that has a bacterial, viral and/or fungalinfection). In some embodiments, the subject to be treated is one thathas a greater than normal risk of being exposed to an agent or material(e.g., a toxin or toxins). In some embodiments, the subject is asoldier, an emergency responder or other subject that has a higher thannormal risk of being exposed to a toxin (e.g., biological toxin),wherein treatment with the compositions and/or methods of the inventionprovide the subject one or more immune response benefits (e.g.,administration of an immunotherapeutic composition to a soldier preventsthe soldier from showing signs or symptoms of disease or morbiditynormally associated with exposure to a toxin).

The invention thus provides methods and compositions for preventingand/or treating infections associated with bacterial, viral, fungal, andyeast microorganisms. In some embodiments, the invention providescompositions (e.g., kits) and methods for identifying subjects (e.g.,subjects vaccinated with one or more bacterial, viral and/or fungalmicrobial antigens/vaccines) useful for providing donor plasma/serum(e.g., with high titers of bacterial, viral and/or fungal-specificantibodies). In some embodiments, the invention provides new therapeuticcompositions for active and passive immunization against infectionscaused by and/or associated with a microbial (e.g., bacterial, viral,fungal, etc.) pathogens. In some embodiments, the invention provides newtherapeutic compositions for active and passive immunization againstinfections caused by and/or associated with a specific virus (e.g.,respiratory syncytial virus), bacteria (e.g., S. pneumonia) fungus oryeast. The invention is not limited by the type of infection caused byand/or associated with S. pneumoniae. Indeed, compositions and methodsof the invention are useful for any and all infections associated withthe presence of S. pneumonia (e.g., including, but not limited to,pneumoniae, bacteraemia, meningitis, otitis media, etc.). For example,in some embodiments, compositions and methods of the invention areutilized with (e.g., administered to) subjects with an immunedeficiency. As described in detail herein, the invention is not limitedto any particular immune deficiency. An immune deficiency may becongenital, acquired or the result of immunosuppressive treatment. Insome embodiments, the invention provides a therapeutic composition thatprovides antibodies against microbial infection (e.g., caused by S.pneumonia), increases the rate of opsonization and phagocytosis ofmicrobial pathogens (e.g., S. pneumonia), and/or induces enhancedintracellular killing of microbial pathogens (e.g., S. pneumonia) (e.g.,thereby preventing or clearing microbial infection (e.g., S. pneumoniaeinfection)). In some embodiments, the invention provides animmunological serum against a bacteria, virus, yeast and/or fungi (e.g.,S. pneumonia). In some embodiments, the invention provides plasma/serumthat provides humoral and/or cellular immunity against a bacteria,virus, yeast and/or fungi (e.g., S. pneumonia). In some embodiments, thehumoral and/or cellular immunity is short lived (e.g., 5 weeks, 4 weeks,3 weeks, 2 weeks, or less). In some embodiments, the humoral and/orcellular immunity lasts 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15, 16 or moreweeks.

In some embodiments, plasma (e.g., a plasma pool (e.g., obtained from adonor or plurality of donors (e.g., one or more donors vaccinated withthe same or different microbial antigen (e.g., virus, bacterium and/orfungus)))) is prepared or selected that has a high titer of antibodiesto a specific microorganism (e.g., a virus, bacterium and/or fungus). Insome embodiments, plasma (e.g., a plasma pool (e.g., obtained from adonor or plurality of donors (e.g., one or more donors vaccinated withthe same or different microbial antigen (e.g., virus, bacterium and/orfungus antigen)))) is prepared or selected that has a high titer ofantibodies to two or more microorganisms (e.g., two or more specificviruses, bacteria and/or fungi). In some embodiments, plasma (e.g., aplasma pool (e.g., obtained from a donor or plurality of donors (e.g.,one or more donors vaccinated with the same or different microbialantigen (e.g., virus, bacterium and/or fungus antigen)))) is prepared orselected that has a high titer of antibodies specific for 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or moredifferent microbial antigens (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more different, specificviruses, bacteria and/or fungi (e.g., serotypes of S. pneumonia

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows scatter plots of titers to RSV and titers to Flu A, Flu B,PIV 1 and PIV 3 in Linear Scale

FIG. 2 shows scatter plots of titers to RSV and titers to Flu A, Flu B,PIV 1 and PIV 3 in Log 2 Scale

FIG. 3 depicts graphs indicating the level of neutralizing antibodytiters for respiratory pathogens present in IVIG compositions generatedusing compositions and methods of the invention.

FIG. 4 shows a schematic of a cotton rat model utilized to study thetherapeutic potential of an immunoglobulin prepared as described inExample 2.

FIG. 5 shows a schematic of a cotton rat model utilized to study theprophylactic potential of an immunoglobulin prepared as described inExample 2.

FIG. 6 shows Total White Blood Cell counts and total Lymphocyte countswere reduced in all cyclophosphamide treated animals (groups A, B, C,and G) compared to normal, unmanipulated cotton rats (groups D, E, andF).

FIG. 7 shows lungs of RSV infected immunosuppressed animals displayedincreased epithelial damage compared to the lungs of RSV-infected normalcotton rats (group A compared to group D) and that treatment of animalswith IVIG of the invention (groups B and C) resulted in reduction ofepithelial damage.

FIG. 8 shows viral titers from total lung and nose homogenates measuredon days 4 and 10 post-intranasal challenge with approximately 5.0 Log₁₀of RSV/A/Long in the immunosuppressed cotton rat with and withouttreatment using IVIG of the invention.

FIG. 9 shows RSV gene expression (NS1 mRNA) quantified by qPCR assay inthe lung, liver, and kidney samples collected from group A, B, C, and Danimals on day 10 p.i. The level of RSV transcript was lowest in group Dand highest in group A samples. Treatment of immunosuppressed animalswith IVIG of the invention resulted in statistically-significantreduction in the lung RSV NS1 mRNA level (groups B and C compared togroup A).

FIG. 10A through FIG. 10F show Total White Blood Cell counts and totalLymphocyte counts were reduced in all cyclophosphamide treated animalscompared to normal, unmanipulated cotton rats.

FIG. 11 shows the level of total IgG in the serum ofcyclophosphamide-treated cotton rats was reduced compared to serum ofcontrol animals

FIG. 12 shows viral titers from total lung homogenates measured on days4 and 10 post-intranasal challenge with approximately 5.0 Log₁₀ ofRSV/A/Long.

FIG. 13 shows RSV gene expression (NS1 mRNA) quantified by qPCR assay inthe lung, kidney and liver of one animal from Group A (animal #96998)and one animal from Group B (animal #97009). NS1 mRNA expression wasdetected in all three organs collected from the animal in Group A, andin the lung and liver of animal in Group B.

DEFINITIONS

As used herein, the term “subject” refers to any human or animal (e.g.,non-human primate, rodent, feline, canine, bovine, porcine, equine,etc.).

As used herein, the term “sample” is used in its broadest sense andencompass materials obtained from any source. As used herein, the term“sample” is used to refer to materials obtained from a biologicalsource, for example, obtained from animals (including humans), andencompasses any fluids, solids and tissues. In particular embodiments ofthis invention, biological samples include blood and blood products suchas plasma, serum and the like. However, these examples are not to beconstrued as limiting the types of samples that find use with thepresent invention.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat is typically composed of two identical pairs of polypeptide chains,each pair having one “light” (L) chain and one “heavy” (H) chain. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 3 or more amino acids. Eachheavy chain is comprised of a heavy chain variable region (abbreviatedherein as HCVR or V_(H)) and a heavy chain constant region. The heavychain constant region is comprised of three domains, C_(H1), C_(H2) andC_(H3). Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region is comprised of one domain, CL. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy/lightchain pair (V_(H) and V_(L)), respectively, form the antibody bindingsite. The term “antibody” encompasses an antibody that is part of anantibody multimer (a multimeric form of antibodies), such as dimers,trimers, or higher-order multimers of monomeric antibodies. It alsoencompasses an antibody that is linked or attached to, or otherwisephysically or functionally associated with, a non-antibody moiety.Further, the term “antibody” is not limited by any particular method ofproducing the antibody. For example, it includes, inter alia,recombinant antibodies, synthetic antibodies, monoclonal antibodies,polyclonal antibodies, bi-specific antibodies, and multi-specificantibodies.

As used herein, the term “antibody derivative” or “derivative” of anantibody refers to a molecule that is capable of binding to the sameantigen that the antibody from which it is derived binds to andcomprises an amino acid sequence that is the same or similar to theantibody linked to an additional molecular entity. The amino acidsequence of the antibody that is contained in the antibody derivativemay be the full-length antibody, or may be any portion or portions of afull-length antibody. The additional molecular entity may be a chemicalor biological molecule. Examples of additional molecular entitiesinclude chemical groups, amino acids, peptides, proteins (such asenzymes, antibodies), and chemical compounds. The additional molecularentity may have any utility, such as for use as a detection agent,label, marker, pharmaceutical or therapeutic agent. The amino acidsequence of an antibody may be attached or linked to the additionalentity by chemical coupling, genetic fusion, noncovalent association orotherwise. The term “antibody derivative” also encompasses chimericantibodies, humanized antibodies, and molecules that are derived frommodifications of the amino acid sequences of an antibody, such asconservation amino acid substitutions, additions, and insertions.

As used herein, the term “antigen” refers to any substance that iscapable of inducing an adaptive immune response. An antigen may be wholecell (e.g. bacterial cell), virus, fungus, or an antigenic portion orcomponent thereof. Examples of antigens include, but are not limited to,microbial pathogens, bacteria, viruses, proteins, glycoproteins,lipoproteins, peptides, glycopeptides, lipopeptides, toxoids,carbohydrates, tumor-specific antigens, and antigenic portions orcomponents thereof.

As used herein, the term “antigen-binding fragment” of an antibodyrefers to one or more portions of a full-length antibody that retain theability to bind to the same antigen that the antibody binds to.

As used herein, the terms “immunoglobulin,” “immunoglobulin molecule”and “IG” encompass (1) antibodies, (2) antigen-binding fragments of anantibody, and (3) derivatives of an antibody, each as defined herein. Asdescribed herein, immunoglobulin may be prepared from (e.g.,fractionated from, isolated from, purified from, concentrated from,etc.) pooled plasma compositions (e.g., for administration to asubject). As used herein, the term “Intravenous immunoglobulin (IVIG)”refers to conventional immunoglobulin prepared from the plasma of overone thousand random human donors, whereas the term “IVIG of theinvention,” for example RSV-IVIG described herein, and in particular, inthe Examples, refers to immune globulin prepared from one thousand ormore human donors, according to methods of the invention, that containsan elevated RSV specific antibody titer and an elevated antibody titerfor one or more other respiratory pathogens (e.g., parainfluenza virus1, parainfluenza virus 2, coronavirus OC43 and/or coronavirus 229E)compared to a control sample (e.g., conventional IVIG prepared from amixture of plasma samples obtained from 1000 or more random human plasmadonors). As used herein, the terms “hyperimmune globulin,” “hyperimmuneserum globulin” and “hyperimmune immune globulin” refer to immune serumglobulin having a high titer of antibodies specific for a singleorganism or antigen (e.g., specific for hepatitis, specific for tetanus,specific for rabies, or specific for varicella zoster) produced fromplasma or serum obtained from a donor(s) that has an elevated antibodytiter for the single, specific organism or antigen. For example,Varicella Zoster Immune Globulin (VZIG, Massachusetts Public HealthBiologic Laboratories, Boston, Mass.; or VARIZIG, Cangene Corporation,Winnipeg, Canada)) is a purified human immune globulin that has a highantibody titer specific for varicella zoster prepared from severalhundred plasma donors and lacks significant antibody titers, or hasdecreased antibody titers, for other organisms or antigens (e.g.,measles). Other hyperimmune globulin products are generally producedfrom donors that have been immunized to the specific pathogen or antigen(e.g., Rabies Immune Globulin, HYPERRAB, Grifols, Clayton, N.C.,produced from a few hundred or less donors immunized with rabiesvaccine). Commercially available hyperimmune globulin products do notmeet FDA specifications for infusion into certain patient populations(e.g., immune deficient patients).

As used herein, the term “antibody sample” refers to anantibody-containing composition (e.g., fluid (e.g., plasma, blood,purified antibodies, blood or plasma fractions, blood or plasmacomponents etc.)) taken from or provided by a donor (e.g., naturalsource) or obtained from a synthetic, recombinant, other in vitrosource, or from a commercial source. The antibody sample may exhibitelevated titer of a particular antibody or set of antibodies based onthe pathogenic/antigenic exposures (e.g., natural exposure or throughvaccination) of the donor or the antibodies engineered to be produced inthe synthetic, recombinant, or in vitro context. Herein, an antibodysample with elevated titer of antibody X is referred to as an“X-elevated antibody sample.” For example, an antibody sample withelevated titer of antibodies against cytomegalovirus is referred to as a“cytomegalovirus-elevated antibody sample.”

As used herein, the term “isolated antibody” or “isolated bindingmolecule” refers to an antibody or binding molecule that is identifiedand separated from at least one contaminant with which it is ordinarilyassociated in its source. Examples of an isolated antibody include: anantibody that: (1) is not associated with one or more naturallyassociated components that accompany it in its natural state; (2) issubstantially free of other proteins from its origin source; or (3) isexpressed recombinantly, in vitro, or cell-free, or is producedsynthetically and the is removed the environment in which it wasproduced.

As used herein, the terms “pooled plasma,” “pooled plasma samples” and“pooled plasma composition” refer to a mixture of two or more plasmasamples and/or a composition prepared from same (e.g., immunoglobulin).Elevated titer of a particular antibody or set of antibodies in pooledplasma reflects the elevated titers of the antibody samples that make upthe pooled plasma. For example, plasma samples may be obtained fromsubjects that have been vaccinated (e.g., with a vaccine) or that havenaturally high titers of antibodies to one or more pathogens as comparedto the antibody level(s) found in the population as a whole. Uponpooling of the plasma samples, a pooled plasma composition is produced(e.g., that has elevated titer of antibodies specific to a particularpathogen). Herein, a pooled plasma with elevated titer of antibody X(e.g., wherein “X” is a microbial pathogen) is referred to as“X-elevated antibody pool.” For example, a pooled plasma with elevatedtiter of antibodies against cytomegalovirus is referred to as“cytomegalovirus-elevated antibody pool.” Also used herein is the term“primary antibody pool” which refers to a mixture of two or more plasmasamples. Elevated titer of a particular antibody or set of antibodies ina primary antibody pool reflects the elevated titers of the antibodysamples that make up the primary antibody pool. For example, many plasmadonations may be obtained from subjects that have been vaccinated (e.g.,with a polyvalent Pseudomonas aeruginosa vaccine). Upon pooling of theplasma samples, a primary antibody pool is produced that has elevatedtiter of antibodies to Pseudomonas aeruginosa. Herein, a primaryantibody pool with elevated titer of antibody X (e.g., wherein “X” is amicrobial pathogen) is referred to as “X-elevated antibody pool.” Forexample, a primary antibody pool with elevated titer of antibodiesagainst cytomegalovirus is referred to as “cytomegalovirus-elevatedantibody pool.” Pooled plasma compositions can be used to prepareimmunoglobulin (e.g., that is subsequently administered to a subject)via methods known in the art (e.g., fractionation, purification,isolation, etc.). The invention provides that both pooled plasmacompositions and immunoglobulin prepared from same may be administeredto a subject to provide prophylactic and/or therapeutic benefits to thesubject. Accordingly, the term pooled plasma composition may refer toimmunoglobulin prepared from pooled plasma/pooled plasma samples.

As used herein, the term “secondary antibody pool” or “tailored antibodypool” refer to a mixture of two or more primary antibody pools. Such apool for example, may be tailored to exhibit elevated titer of specificantibodies or sets of antibodies by combining primary pools that exhibitsuch elevated titers. For example, a primary pool with elevated titer ofPseudomonas aeruginosa antibodies could be combined with a primary poolwith elevated titer of Varicella-zoster virus antibodies to produce atailored antibody pool with elevated titer of antibodies againstPseudomonas aeruginosa and Varicella-zoster virus.

As used herein, the term, “spiked antibody pool” refers to a pooledplasma sample (e.g., primary or tailored antibody pool) that containsantibodies from at least one natural source spiked or combined withantibodies or other immunoglobulin produced synthetically,recombinantly, or through other in vitro means.

As used herein, the term “isolated antibody” or “isolated bindingmolecule” refers to an antibody or binding molecule that is identifiedand separated from at least one contaminant with which it is ordinarilyassociated in its source. Examples of an isolated antibody include: anantibody that: (1) is not associated with one or more naturallyassociated components that accompany it in its natural state; (2) issubstantially free of other proteins from its origin source; or (3) isexpressed recombinantly, in vitro, or cell-free, or is producedsynthetically and the is removed the environment in which it wasproduced.

As used herein, the term “purified” or “to purify” means the result ofany process that removes some of a contaminant from the component ofinterest, such as a protein (e.g., antibody) or nucleic acid. Thepercent of a purified component is thereby increased in the sample.

As used herein, the term “immunotherapeutic agents” refers to a chemicalor biological substance that can enhance an immune response (e.g.,specific or general) of a mammal.

Examples of immunotherapeutic agents include: passively administeredprimary antibody pools; tailored antibody pools (e.g., passivelyadministered tailored antibody pools); vaccines, chemokines, antibodies,antibody fragments, bacillus Calmette-Guerin (BCG); cytokines such asinterferons; vaccines such as MyVax personalized immunotherapy,Onyvax-P, Oncophage, GRNVAC1, FavId, Provenge, GVAX, Lovaxin C,BiovaxID, GMXX, and NeuVax; and antibodies such as alemtuzumab(CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX), gemtuzunabozogamicin (MYLOTARG), ibritumomab tiuxetan (ZEVALIN), panitumumab(VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN),tositumomab (BEXXAR), tremelimumab, CAT-3888, agonist antibodies to CD40receptor that are disclosed in WO2003/040170, and any immunomodulatingsubstance.

As used herein, the term “donor” refers to a subject that provides abiological sample (e.g., blood, plasma, etc.). A donor/donor sample maybe screened for the presence or absence of specific pathogens (e.g.,using U.S. Food and Drug Administration (FDA) guidelines for assessingsafety standards for blood products (e.g., issued by the FDA BloodProducts Advisory Committee). For example, a donor/donor sample may bescreened according to FDA guidelines to verify the absence of one ormore bloodborne pathogens (e.g., human immunodeficiency virus (HIV) 1(HIV-1), HIV-2; Treponema pallidum (syphilis); Plasmodium falciparum, P.malariae, P. ovale, P. vivax or P. knowlesi (malaria); hepatitis Bvirus(HBV), hepatitis C virus HCV); prions (Creutzfeldt Jakob disease);West Nile virus; parvovirus; Typanosoma cruzi; SARS coronavirus (SARS);vaccinia virus or other pathogen routinely screened or that isrecommended to be screed for by a regulatory body such as the FDA). Asused herein, the terms “selected donor,” “selected human subject” andthe like refer to a subject that is chosen and/or identified to providea biological sample (e.g., blood, plasma, etc.) based on the presence ofa desired characteristic of that biological sample (e.g., a specifictiter (e.g., high, average or low titer) of antibodies (e.g., determinedusing one or more screening methods (e.g., neutralization assay or otherassay described herein) specific for one or more pathogens (e.g., one ormore respiratory pathogens (e.g., respiratory syncytial virus))). Forexample, in one embodiment described herein, a high titer selected donor(e.g., identified by characterizing the specific titer of antibodies torespiratory syncytial virus via a first, plasma screening assay utilizedto assess RSV neutralizing activity in a donor plasma sample; and asecond screening assay characterizing the specific antibody titer of thepurified immunoglobulin fraction of each donor plasma sample identifiedas displaying the top 20% of neutralizing activity using the first,plasma screening assay, wherein a purified immunoglobulin fractionpossessing an RSV neutralization titer of 1800 or above is used tocategorize a plasma sample as a high titer selected donor) comprises apathogen specific antibody titer that is about 1.5-2.0 times, 2-5 times,5-8 times, 8-10 times, 10-14 times, 14 times or greater than a standardvalue (the titer of pathogen specific antibodies present in a pool ofplasma samples from 1000 or more random human subjects), wherein mediumtiter donors comprise a pathogen specific antibody titer that is thetiter of pathogen specific antibodies present in a pool of plasmasamples from 1000 or more random human subjects or that is onlymarginally higher (e.g., 5-20% higher) or marginally lower (e.g., 5-20%lower) than this value, and wherein low titer donors comprise a pathogenspecific antibody titer that is around 20-50 percent the titer ofpathogen specific antibodies present in a pool of plasma samples from1000 or more random human subjects. As used herein, a “non-selecteddonor,” “random donor,” “random human subject” and the like, when usedin reference to a donor sample (e.g., blood, plasma, etc.) used forgenerating a pool of donor samples), refer to a subject that provides abiological sample (e.g., blood, plasma, etc.) without specific knowledgeof characteristics (e.g., antibody titer to one or more pathogens) ofthat sample. Thus, a random donor/random donor sample may be asubject/sample that passes FDA bloodborne pathogen screeningrequirements and is not selected on the basis of antibody titers (e.g.,respiratory pathogen specific antibody titers). In one embodimentdescribed herein, the titer for non-tested/non-selected sourcedonor/donor sample is set at zero. If biological samples from a group ofselected donors selected for the same characteristic are pooled, thepool so generated (e.g., a primary pool) will be enhanced for theselected characteristic. On the other hand, if biological samples from agroup of non-selected, random donors are pooled, random differencesbetween the biological samples will be averaged out, and the pool sogenerated (e.g., the primary pool) will not be enhanced for any specificcharacteristic. It is preferred that both random donors/random donorsamples and selected donors/selected donor samples are screened (e.g.,using FDA screening requirements) to verify the absence of bloodbornepathogens (e.g., prior to and/or after pooling). Furthermore, accordingto one embodiment of the invention, and as described in detail herein,biological samples (e.g., plasma samples) from one or more selecteddonors can be mixed with biological samples (e.g., plasma samples) fromone or more other selected donors (e.g., selected for the same ordifferent characteristic (e.g., the same or different titer (e.g., high,medium or low titer) of antibodies to a specific pathogen) and/or mixedwith biological samples (e.g., plasma samples) from one or morenon-selected donors in order to generate a pooled plasma composition(e.g., that contains a desired, standardized level of antibodies for oneor more specific pathogens (e.g., one or more respiratory pathogens)).

As used herein, an “immunostimulatory amount” refers to that amount of avaccine (e.g., viral, bacterial and/or fungal vaccine) that is able tostimulate the immune response. An immune response includes the set ofbiological effects leading to the body's production of immunoglobulins,or antibodies, in response to a foreign entity. Accordingly, immuneresponse refers to the activation of B cells, in vivo or in culture,through stimulation of B cell surface Ig receptor molecules. Themeasurement of the immune response is within the ordinary skill of thosein this art and includes the determination of antibody levels usingmethods described in the series by P. Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology: Practice and Theory of EnzymeImmunoassays, (Burdon & van Knippenberg eds., 3rd ed., 1985) Elsevier,New York; and Antibodies: A Laboratory Manual, (Harlow & Lane eds.,1988), Cold Spring Harbor Laboratory Press; as well as procedures suchas countercurrent immuno-electrophoresis (GIEP), radioimmunoassay,radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530, all of which are incorporated by reference. Measurement ofthe immune response also includes detection or determination of B cellactivation events that may precede antibody production, or signal anincrease in antibody production. Such measurements include, B cellproliferation assays, phosphorylation assays, assays of intracytoplasmicfree calcium concentration, and other methods of determining B cellactivation known in the art. Representative assays are provided inMongini et al., J. Immunol. 159:3782-91 (1997); Frade, et al., BBRC188:833-842 (1992); Tsokos et al., J. Immunol. 144:1640-1645 (1990);Delcayre et al., BBRC 159:1213-1220 (1989); and Nemerow et al., J.Immunol. 135:3068-73 (1985) each of which is incorporated by reference.In preferred embodiments, the practice of the invention includespromoting, enhancing or stimulating an immune response. These actionsrefer to establishing an immune response that did not previously exist;to optimizing or increasing a desired immune response; to establishingor increasing a secondary response characterized by increased isotypeswitching, memory response, or both; to providing a statisticallyincreased immunoprotective effect against a pathogen; to generating anequivalent or greater humoral immune response, or other measure of Bcell activation, from a reduced or limiting dose of antigen; togenerating an increased humoral immune response, or other measure of Bcell activation, in response to an equivalent dose of antigen; or tolowering the affinity threshold for B cell activation in vivo or invitro. Preferably, an immunostimulatory amount refers to that amount ofvaccine that is able to stimulate an immune response in a subject (e.g.,a donor), and from which subject plasma, serum or other blood componentis harvested for use in the compositions and methods of the invention(e.g., for the therapeutic and/or prophylactic treatment of microbial(e.g., viral, bacterial and/or fungal) infection in a subject treatedwith compositions and methods described herein)).

The terms “buffer” or “buffering agents” refer to materials, that whenadded to a solution, cause the solution to resist changes in pH.

The terms “reducing agent” and “electron donor” refer to a material thatdonates electrons to a second material to reduce the oxidation state ofone or more of the second material's atoms.

The term “monovalent salt” refers to any salt in which the metal (e.g.,Na, K, or Li) has a net 1+ charge in solution (i.e., one more protonthan electron).

The term “divalent salt” refers to any salt in which a metal (e.g., Mg,Ca, or Sr) has a net 2+ charge in solution.

The terms “chelator” or “chelating agent” refer to any materials havingmore than one atom with a lone pair of electrons that are available tobond to a metal ion.

The term “solution” refers to an aqueous or non-aqueous mixture.

As used herein, the term “adjuvant” refers to any substance that canstimulate an immune response (e.g., a mucosal immune response). Someadjuvants can cause activation of a cell of the immune system (e.g., anadjuvant can cause an immune cell to produce and secrete a cytokine).Examples of adjuvants that can cause activation of a cell of the immunesystem include, but are not limited to, the nanoemulsion formulationsdescribed herein, saponins purified from the bark of the Q. saponariatree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLCfractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); cholera toxin (CT), and Leishmaniaelongation factor (a purified Leishmania protein; Corixa Corporation,Seattle, Wash.). Traditional adjuvants are well known in the art andinclude, for example, aluminum phosphate or hydroxide salts (“alum”). Insome embodiments, compositions of the present invention are administeredwith one or more adjuvants (e.g., to skew the immune response towards aTh1 and/or Th2 type response). In some embodiments, an adjuvantsdescribed in US2005158329; US2009010964; US2004047882; or U.S. Pat. No.6,262,029 (each of which is hereby incorporated by reference in itsentirety) is utilized.

As used herein, the term “an amount effective to induce an immuneresponse” (e.g., of a composition for inducing an immune response),refers to the dosage level required (e.g., when administered to asubject) to stimulate, generate and/or elicit an immune response in thesubject. An effective amount can be administered in one or moreadministrations (e.g., via the same or different route), applications ordosages and is not intended to be limited to a particular formulation oradministration route.

As used herein, the term “under conditions such that said subjectgenerates an immune response” refers to any qualitative or quantitativeinduction, generation, and/or stimulation of an immune response (e.g.,innate or acquired).

A used herein, the term “immune response” refers to a response by theimmune system of a subject. For example, immune responses include, butare not limited to, a detectable alteration (e.g., increase) inToll-like receptor (TLR) activation, lymphokine (e.g., cytokine (e.g.,Th1 or Th2 type cytokines) or chemokine) expression and/or secretion,macrophage activation, dendritic cell activation, T cell activation(e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cellactivation (e.g., antibody generation and/or secretion). Additionalexamples of immune responses include binding of an immunogen (e.g.,antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducinga cytotoxic T lymphocyte (“CTL”) response, inducing a B cell response(e.g., antibody production), and/or T-helper lymphocyte response, and/ora delayed type hypersensitivity (DTH) response against the antigen fromwhich the immunogenic polypeptide is derived, expansion (e.g., growth ofa population of cells) of cells of the immune system (e.g., T cells, Bcells (e.g., of any stage of development (e.g., plasma cells), andincreased processing and presentation of antigen by antigen presentingcells. An immune response may be to immunogens that the subject's immunesystem recognizes as foreign (e.g., non-self antigens frommicroorganisms (e.g., pathogens), or self-antigens recognized asforeign). Thus, it is to be understood that, as used herein, “immuneresponse” refers to any type of immune response, including, but notlimited to, innate immune responses (e.g., activation of Toll receptorsignaling cascade) cell-mediated immune responses (e.g., responsesmediated by T cells (e.g., antigen-specific T cells) and non-specificcells of the immune system) and humoral immune responses (e.g.,responses mediated by B cells (e.g., via generation and secretion ofantibodies into the plasma, lymph, and/or tissue fluids). The term“immune response” is meant to encompass all aspects of the capability ofa subject's immune system to respond to antigens and/or immunogens(e.g., both the initial response to an immunogen (e.g., a pathogen) aswell as acquired (e.g., memory) responses that are a result of anadaptive immune response).

As used herein, the terms “immunogen” and “antigen” refer to an agent(e.g., a microorganism (e.g., bacterium, virus or fungus) and/or portionor component thereof (e.g., a protein antigen or a polysaccharide)) thatis capable of eliciting an immune response in a subject.

As used herein, the term Streptococcus (e.g., S. pneumoniae) antigenrefers to a component or product of a bacteria of the genusStreptococcus that elicits an immune response when administered to asubject.

As used herein, the term “pathogen product” refers to any component orproduct derived from a pathogen including, but not limited to,polypeptides, peptides, proteins, nucleic acids, membrane fractions, andpolysaccharides.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions (e.g., toxic, allergic orimmunological reactions) when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, and various types ofwetting agents (e.g., sodium lauryl sulfate), any and all solvents,dispersion media, coatings, sodium lauryl sulfate, isotonic andabsorption delaying agents, disintrigrants (e.g., potato starch orsodium starch glycolate), polyethyl glycol, other natural andnon-naturally occurring carries, and the like. The compositions also caninclude stabilizers and preservatives. Examples of carriers, stabilizersand adjuvants have been described and are known in the art (See e.g.,Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co.,Easton, Pa. (1975), incorporated herein by reference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acomposition of the present invention that is physiologically toleratedin the target subject. “Salts” of the compositions of the presentinvention may be derived from inorganic or organic acids and bases.Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compositions of the inventionand their pharmaceutically acceptable acid addition salts. Examples ofbases include, but are not limited to, alkali metal (e.g., sodium)hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia,and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, and the like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

For therapeutic use, salts of the compositions of the present inventionare contemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable composition.

As used herein, the terms “at risk for infection” and “at risk fordisease” refer to a subject that is predisposed to experiencing aparticular infection or disease (e.g., respiratory infection ordisease). This predisposition may be genetic (e.g., a particular genetictendency to experience the disease, such as heritable disorders), or dueto other factors (e.g., immunosuppression, compromised immune system,immunodeficiency, environmental conditions, exposures to detrimentalcompounds present in the environment, etc.). Thus, it is not intendedthat the present invention be limited to any particular risk (e.g., asubject may be “at risk for disease” simply by being exposed to andinteracting with other people), nor is it intended that the presentinvention be limited to any particular disease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for thetreatment of immunodeficiency (e.g., primary immunodeficiency disease).In particular, the invention provides pooled human plasma compositionsand/or immunoglobulin prepared from same, methods of identifying humanplasma for use in the compositions, methods of manufacturing (e.g.,utilizing pooling and blending methods described herein) thecompositions, and methods of utilizing the compositions (e.g., forprophylactic administration and/or therapeutic treatment (e.g., passiveimmunization (e.g., immune-prophylaxis))).

Immunoglobulin obtained from the plasma of thousands of different donorscontains antibodies to many of the pathogens that these individuals haveencountered in their lifetime. However, a significant limitation exists.Since immunoglobulin is pooled from thousands of donors the antibodytiters to the many infectious organisms (e.g., microbial pathogens) forwhich protection is sought varies greatly and is very often notsufficient to meet the immune needs in case of an infection (e.g., witha pathogen) in an immune suppressed individual.

Hyperimmune serum globulins (immune serum globulin having high titers ofa particular antibody), in distinction to normal immunoglobulin, havebeen therapeutically useful in treating patients who require immediateinfusion of high titer antibodies. For example, tetanus hyperimmuneglobulin is useful in treating patients who may have suspected tetanusand rabies hyperimmune globulin for treating patients with suspectedrabies. Hyperimmune serum globulins can be produced from plasma or serumobtained from a selected donor(s) who have elevated titers for aspecific antibody than is normally found in the average population (thatis not found at a high titer in the average population). These donorshave either been recently immunized with a particular vaccine (See,e.g., U.S. Pat. No. 4,174,388) or else they have recently recovered froman infection or disease (See, e.g., Stiehm, Pediatrics, Vol. 63, No. 1,301-319 (1979); herein incorporated by reference in its entirety). Thesehigh titer sera or plasmas are pooled and subjected to fractionationprocedures (Cohn et al, J. Am. Chem. Soc., 68, 459 (1946); Oncley, etal, J. Am. Chem. Soc., 71, 541 (1949); herein incorporated by referencein their entireties). Such procedures have required specific selectionof a donor or limited numbers of donors in order to produce hyperimmuneglobulin with elevated concentrations of the desired antibodies.

Many different microorganisms are commonly found in the human upperrespiratory system. S. pneumoniae is one such example, While S.pneumoniae is part of the normal upper respiratory tract flora, as withmany natural flora, it can become pathogenic under certain conditions(e.g., if the immune system of the host is suppressed). This is the casewith other infections as well that become virulent in immune suppressedhosts.

Cytomegalovirus (CMV) is a genus of viruses, some of which have thepotential to infect humans and cause disease. While infection is notcommon among the general population, it is encountered very frequentlyin certain susceptible groups of patients. Immunosuppressed organtransplant and cancer patients have been identified as having anunusually high risk of acquiring severe, and sometimes fatal, CMVinfection.

Respiratory syncytial virus (RSV) is considered the most important causeof severe respiratory disease in infants and young children. It can alsobe an important cause of lower respiratory tract disease in the elderly,hematopoietic stem cell transplant patients and organ transplantpatients. In the United States alone it has been reported that thisvirus causes pneumonia, bronchitis and croup in approximately 4 millionchildren each year, resulting in about 4500 deaths. In the western worldit is the major cause for hospitalization of children (National ResearchCouncil News Report, 35, 9 (1985); Stott, E. J. et al, Archives ofVirology, 84:1-52 (1985); and W. H. O. Scientific Group, World HealthOrganization Technical Report Series 642 (1980); herein incorporated byreference in their entireties).

Varicella-zoster virus (VZV) is the cause of clinical disease that,although not common among the general population, is encounteredfrequently in certain susceptible groups of patients. Immunosuppressedorgan transplant and cancer patients have been identified as having anunusually high risk of acquiring severe, and frequently fatal, VZVinfection. Zaia et al in The Journal of Infectious Diseases, Vol. 137,No. 5, 601-604 (1978) disclosed a practical method for preparation ofVZV immune globulin for intramuscular administration. Outdated blood wasscreened for complement-fixing antibody to VZV. About 15% of the plasmaunits had a complement-fixation titer equal to or greater than 1:16,with about 7.5% greater than or equal to 1:32.

Pseudomonas aeruginosa (P. aeruginosa) is a common bacterium that cancause disease in animals, including humans. Although infection with P.aeruginosa is not common among the general population, P. aeruginosainfection is encountered very frequently in certain susceptible groupsof patients. Burn victims, immunosuppressed cancer patients, andindividuals with extended hospital stays have been identified as havingan unusually high risk of acquiring severe, and sometimes fatal diseasecaused by P. aeruginosa. James et al, in The Lancet, 13 Dec. 1980,1263-1265 (herein incorporated by reference in its entirety), describedpassive immunization of burn patients at risk of septicaemia. Theimmunization was accomplished with an immunoglobulin prepared fromplasma from healthy human volunteers vaccinated with a polyvalentPseudomonas vaccine.

The present invention relates to compositions and methods for thetreatment of immunodeficiency (e.g., primary immunodeficiency disease).In particular, the invention provides pooled human plasma immunoglobulincompositions, methods of identifying human plasma for use in thecompositions, methods of manufacturing the compositions, and methods ofutilizing the compositions (e.g., for prophylactic administration and/ortherapeutic treatment (e.g., passive immunization (e.g.,immune-prophylaxis))). In one embodiment, the invention provides acomposition comprising pooled plasma samples obtained from 1000 or morehuman subjects, wherein the pooled plasma comprises elevated levels,compared to the pathogen-specific antibody titers found in a mixture ofplasma samples obtained from 1000 or more random human subjects, ofpathogen-specific antibody titers to one or more respiratory pathogens.The invention is not limited by the type of respiratory pathogens forwhich the pooled plasma comprises elevated levels of pathogen-specificantibody titers. The pooled plasma composition may comprise elevatedlevels of pathogen-specific antibody titers to one or more ofrespiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirus,coronavirus, S. pneumonia, H. influenza, L. pneumophila, group AStreptococcus, or any other respiratory pathogen known by those ofordinary skill in the art or described herein. In certain embodiments,the invention provides a composition comprising pooled plasma samplesfor passive immunization of patients susceptible to, or suffering from,infection (e.g., bacterial infection (e.g., associated withStreptococcus pneumonia, Haemophilus influenza, etc.), viral infection(e.g., associated with adenovirus, herpes virus, influenza virus,rhinovirus, etc.), fungal infection (e.g., associated with Aspergillusfumigatus, Cladosporium or other fungi) and/or exposure to microbialtoxins and/or harmful agents (e.g., animal venom))).

Accordingly, disclosed herein are methods and compositions for thepassive immunization of subjects infected with or who are susceptible toinfection, disease or harm from various pathogens (e.g., pathogenicbacteria (e.g., S. aureus, P. aeruginosa, etc.), pathogenic viruses(e.g., RSV, CMV, etc.), pathogenic eukaryotes, toxins (e.g., bacterialtoxins (e.g., Botulinum neurotoxin, Tetanus toxin, Clostridium difficiletoxin, E. coli toxin, Vibrio RTX toxin, Staphylococcal toxins,Cyanobacteria toxin), fungal toxins, e.g. mycotoxins)), etc. Methodsinclude, for example, the blending and/or pooling of plasma samples togenerate a composition comprising pooled plasma samples (e.g., from 1000or more donor subjects) comprising specific, elevated levels ofpathogen-specific antibody titers and/or immunoglobulin prepared fromsame (e.g., for the treatment of infection wherein administration (e.g.,infusion) of conventional IVIG would not provide sufficient antibodylevels required to treat a specific infection). The pooled plasmacomposition may comprise elevated levels, compared to a control value(e.g., the levels of pathogen-specific antibody titers in 1000 or morerandom donor plasma samples), of pathogen-specific antibody titers toone or more of respiratory syncytial virus, influenza A virus, influenzaB virus, parainfluenza virus type 1, parainfluenza virus type 2,metapneumovirus, coronavirus, S. pneumonia, H. influenza, L.pneumophila, group A Streptococcus, or any other respiratory or othertype of pathogen known by those of ordinary skill in the art ordescribed herein. In one embodiment, donor plasma samples that containhigh antibody titers to a specific respiratory pathogen (e.g., RSV) areidentified, selected and blended with other donor plasma samples that donot contain high antibody titers to a specific respiratory pathogen(e.g., RSV) in order to generate a pool of at least 1000 donor plasmasamples, wherein the pool contains a desired (e.g., standardized,elevated) antibody titer to one or more specific respiratory pathogens(e.g., RSV), in the absence of having to utilize 1000 high titer donors.For example, in some embodiments, the identification, selection andblending processes of the invention allow generation of pooled plasmasamples from at least 1000 donors, wherein the pooled plasma contains adesired (e.g., standardized, elevated) antibody titer to one or morespecific respiratory pathogens (e.g., RSV), wherein less than about 50%(e.g., 50-45%, 45-40%, 40-35%, 35-30%, 30-25%, 25-20% or fewer) of thedonors are identified as high titer for RSV and/or other respiratorypathogen specific antibodies. In one embodiment, a pooled plasmacomposition (e.g., pooled plasma samples or immunoglobulin prepared fromsame) is provided wherein the pooled plasma composition contains plasmafrom 1000 or more donors, wherein about 40-50% of the donor plasmasamples have high titer for RSV and/or other respiratory pathogenspecific antibodies, 20-30% of the donor plasma samples contain a mediumtiter for RSV and/or other respiratory pathogen specific antibodies, and20-40% of the donor plasma samples have low titer for RSV and/or otherrespiratory pathogen specific antibodies. In another embodiment, plasmasamples from 1000 or more (e.g., 1000, 1000-1500, 1500-2000, 2000-3000,3000-4000, 4000-6000, 6000-8000 or more) human subjects arecharacterized for antibody titers (e.g., using one or more assaysdescribed herein (e.g., using a first neutralization or other type ofassay described herein and/or a second neutralization or other type ofassay described herein)) to one or more respiratory viruses selectedfrom respiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirus,coronavirus, S. pneumonia, H. influenza, L. pneumophila, and group AStreptococcus; the subjects are categorized as high, medium or low titerfor the one or more respiratory viruses; plasma samples are obtainedfrom a subset of the subjects identified as high, medium or low titer;and plasma samples from at least 500 (e.g., at least 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more)subjects so identified are blended together in order to generate apooled plasma composition (e.g., comprising a desired, elevated antibodytiter to one or more of respiratory viruses selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus, coronavirus,S. pneumonia, H. influenza, L. pneumophila, and group A Streptococcus).

In one embodiment, identifying antibody titer comprises a first, plasmascreening assay assessing neutralizing activity/titer (e.g., RSVneutralizing activity) in a plasma sample, and a second screening assayassessing antibody titer in a purified immunoglobulin fraction of theplasma sample (e.g., specific RSV antibody titer in the purifiedimmunoglobulin fraction). In one embodiment, neutralizing activity inplasma is measured via the absence of infection of hepatocytes. In afurther embodiment, the first plasma screening assay assessingneutralization activity categorizes plasma samples as high titer, mediumtiter, or low titer for RSV specific antibodies, wherein a high titerRSV donor/donor sample is one having an anti-RSV titer of about 7800 orgreater, a medium titer RSV donor/donor sample is one having an anti-RSVtiter of about 3300-7799, and a low titer RSV donor/donor sample is onehaving an anti-RSV titer of about 1800-3299 (e.g., titer beingcalculated and assigned to a donor/donor sample as the dilution thatgive 50% inhibition of virus growth/infection of hepatocytes (that pointwhich is 50% of the two extremes (saline plus virus is 100 growth and novirus added is 0 growth) according to methods described herein). Instill a further embodiment, only plasma samples identified as displayingthe top 20% of neutralizing activity are processed to produce purifiedimmunoglobulin and subsequently screened using the second screeningassay. In one embodiment, the second screening assay characterizes theRSV specific antibody titer of a purified immunoglobulin fraction of theplasma sample. In a preferred embodiment, a donor/donor sample that hasan RSV neutralization titer of 1800 in a purified immunoglobulinfraction of the plasma sample is scored as a high titer donor. In apreferred embodiment, an RSV neutralization titer of 1800 is used toidentify a donor/donor sample (e.g., plasma sample) comprising elevatedantibody titers for one or more respiratory pathogens selected frominfluenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus and coronavirus compared tothe antibody titer in a control (e.g., the respiratory pathogen specificantibody titer present in a pool of plasma samples from 1000 or morerandom human subjects). In another embodiment, the pooled plasmacomposition comprises an RSV neutralization antibody titer of 1800 ormore.

In one embodiment, when plasma samples are blended from 1000 or moresubjects, less than half (e.g., about 10-20%, 20-30%, 30-40%, 40-50%) ofthe 1000 or more donors are from subjects categorized as high titer(e.g., using the above described first and second screening assays) forthe one or more respiratory viruses (e.g., RSV). For example, in apreferred embodiment, when plasma samples are blended from 1000 or moresubjects, only about 40-50% (e.g., 400-500) of the plasma samples arefrom subjects categorized as high titer (e.g., using the above describedfirst and second screening assays) for the one or more respiratoryviruses (e.g., RSV) and the remainder are characterized as not beinghigh titer (e.g., for the one or more respiratory viruses (e.g., RSV).In another preferred embodiment, when plasma samples are blended from1000 or more subjects, only about 40-45% (e.g., 400-450) of the plasmasamples are from subjects categorized as high titer for the one or morerespiratory viruses. In preferred embodiments, 30-45%, 35-45%, 40-45% ofthe 1000 or more plasma samples are from high titer selected humandonors.

In one embodiment, when plasma samples are blended from 1000 or moresubjects, only about 30-45%, 35-45%, or 30-40% of the plasma samples arefrom subjects categorized as high titer (e.g., using the sequentialfirst and second screening assays described herein) for RSV antibodies,about 55-75% or about 55-65% are from non-high titer selected donors,and about 3-30% or about 3-20% are from non-selected donors.

In one embodiment, less than 50% of the total volume of the pooledplasma composition (e.g., in preferred embodiments, about 30-45%,35-45%, 35-40%)) comprises plasma obtained from high titer selectedhuman donors; about 55-70% (e.g., in preferred embodiment, about 55-75%,55-65%) of the total volume of the pooled plasma composition comprisesplasma obtained from non-high titer selected human donors, and about3-20% of the total volume of the pooled plasma composition comprisesplasma from non-selected human donors.

In one embodiment, when plasma samples are blended from 1000 or moresubjects, the blended plasma or immunoglobulin obtained (e.g.,fractionated) from same contains seroprotective antibody titers tomeasles, diphtheria and polio (e.g., contain antibody titers to measles,diphtheria and polio that provide a subject administered the blendedplasma composition or immunoglobulin obtained from same serum levels ofantibodies specific for measles, diphtheria and polio to prevent, orprotect from, infection with same). In another embodiment, when plasmasamples are blended from 1000 or more subjects, the blended plasma orimmunoglobulin obtained (e.g., fractionated) from same containsseroprotective antibody titers to measles, diphtheria, polio, tetanusand/or varicella (e.g., contain antibody titers to measles, diphtheria,polio, tetanus and/or varicella that provide a subject administered theblended plasma composition or immunoglobulin obtained from same serumlevels of antibodies specific for measles, diphtheria, polio, tetanusand/or varicella to prevent, or protect from, infection with same (e.g.,meets the antibody titer levels recommended by U.S. Food and DrugAdministration (e.g., for the treatment of immune deficiency diseaseand/or treatment of or prevention of infection in an immune deficientsubject))). In another embodiment, the pooled plasma comprises elevatedlevels, compared to the pathogen-specific antibody titers found in amixture of plasma samples obtained from 1000 or more random humansubjects, of pathogen-specific antibody titers to two, three, four ormore respiratory pathogens described herein. In one embodiment, thepooled plasma comprises a respiratory syncytial virus-specific antibodytiter that is at least 2 fold greater (e.g. 2 fold, 3 fold, 4 fold, 5fold 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 12 fold, 15 fold or more)than the respiratory syncytial virus-specific antibody titer found in amixture of plasma samples obtained from 1000 or more random humansubjects. In another embodiment, the pooled plasma comprisespathogen-specific antibody titers to at least two or more respiratorypathogens selected from respiratory syncytial virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus, coronavirus, S. pneumonia, H. influenza, L.pneumophila, and group A Streptococcus that are each elevated at least1.5 fold compared to the pathogen-specific antibody titers found in amixture of plasma samples obtained from 1000 or more random humansubjects. In another embodiment, the pooled plasma comprisespathogen-specific antibody titers to at least three or more respiratorypathogens selected from respiratory syncytial virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus, coronavirus, S. pneumonia, H. influenza, L.pneumophila, and group A Streptococcus that are each elevated at least1.5 fold compared to the pathogen-specific antibody titers found in amixture of plasma samples obtained from 1000 or more random humansubjects. In still another embodiment, the pooled plasma comprisespathogen-specific antibody titers to at least four or more respiratorypathogens selected from respiratory syncytial virus, influenza A virus,influenza B virus, parainfluenza virus type 1, parainfluenza virus type2, metapneumovirus, coronavirus, S. pneumonia, H. influenza, L.pneumophila, and group A Streptococcus that are each elevated at least1.5 fold compared to the pathogen-specific antibody titers found in amixture of plasma samples obtained from 1000 or more random humansubjects. In one embodiment, the pooled plasma comprises plasma samplesobtained from 1000-3000 or more (e.g., more than 1000, 1250, 1500, 1750,2000, 2500, 3000, 3500, 4000 or more human subjects). In one embodiment,the pooled plasma is utilized to prepare immunoglobulin (e.g., forintravenous administration to a subject). In one embodiment, the pooledplasma and/or immunoglobulin provides a therapeutic benefit to a subjectadministered the pooled plasma and/or immunoglobulin that is notachievable via administration of a mixture of plasma samples (orimmunoglobulin prepared from same) obtained from 1000 or more randomhuman subjects. The invention is not limited by the type of therapeuticbenefit provided. Indeed, a variety of therapeutic benefits may beattained including those described herein. In one embodiment, the pooledplasma and/or immunoglobulin possesses enhanced viral neutralizationproperties compared to a mixture of plasma samples obtained from 1000 ormore random human subjects or immunoglobulin prepared from same. Forexample, in one embodiment, the pooled plasma possesses enhanced viralneutralization properties against one or more (e.g., two, three, four,five or more) respiratory pathogens (e.g., described herein). In afurther embodiment, the enhanced viral neutralization properties reduceand/or prevent infection in a subject administered the composition for aduration of time that is longer than, and not achievable in, a subjectadministered a mixture of plasma samples obtained from 1000 or morerandom human subjects. In one embodiment, the pooled plasma and/orimmunoglobulin prepared from same reduces the incidence of infection ina subject administered the composition. In another embodiment, a pooledplasma and/or immunoglobulin prepared from same reduces the number ofdays a subject administered the pooled plasma and/or immunoglobulin isrequired to be administered antibiotics (e.g., to treat infection). Inyet another embodiment, a pooled plasma and/or immunoglobulin preparedfrom same increases the trough level of circulating anti-respiratorypathogen specific antibodies in a subject (e.g., increases the level ofneutralizing titers specific for respiratory pathogens (e.g., therebyproviding protective levels of anti-respiratory pathogen specificantibodies between scheduled dates of administration of the pooledplasma and/or immunoglobulin prepared from same that are not maintainedin a subject administered a mixture of plasma samples obtained from 1000or more random human subjects or immunoglobulin prepared from same)). Inone embodiment, the composition comprising pooled plasma samples furthercomprises a pharmaceutically acceptable carrier (e.g., any natural ornon-naturally occurring carrier(s) known in the art). In one embodiment,a subject administered immunoglobulin prepared from pooled plasmaaccording to the invention displays a mean fold increase in anti-RSVneutralization titer that is at least 4 fold, at least 5 fold, at least6 fold, at least 7 fold, at least 8 fold, at least 9 fold or more at atime point of at least 1 to 14 days post administration (e.g., 14 day,15 days, 16 days, 17 days, 18 days, 19 days or more) of theimmunoglobulin. The invention is not limited by the amount ofimmunoglobulin administered to a subject. In one embodiment, a subjectis administered between 100-5000 mg/kg of the immunoglobulin one time,or daily for two or more days (e.g., 2, 3, 4, or more consecutive days).In another embodiment, such doses are administered intermittently, e.g.every week, every two weeks, every three weeks, every four weeks, etc.In one embodiment, a subject is administered between 750-1500 mg/kg ofimmunoglobulin on day one and between 750-1500 mg/kg immunoglobulin onday 2. In one embodiment, a subject is administered 1500 mg/kg ofimmunoglobulin on day one and 750 mg/kg immunoglobulin on day 2. Inanother embodiment, a subject is administered 750 mg/kg ofimmunoglobulin on day one and 750 mg/kg immunoglobulin on day 2. In oneembodiments, a subject is administered immunoglobulin on day one,optionally administered immunoglobulin on day 2, and thenre-administered immunoglobulin every 21 days. In one embodiments, asubject is administered immunoglobulin on day one, optionallyadministered immunoglobulin on day 2, and then re-administeredimmunoglobulin every 28 days. In one embodiment, the pooled plasmaand/or immunoglobulin prepared from same reduces the incidence ofinfection in a subject administered the composition. In anotherembodiment, a pooled plasma and/or immunoglobulin prepared from samereduces the number of days a subject administered the pooled plasmaand/or immunoglobulin is required to be administered antibiotics (e.g.,to treat infection). In yet another embodiment, a pooled plasma and/orimmunoglobulin prepared from same increases the trough level ofcirculating anti-respiratory pathogen specific antibodies and increasesthe trough level of circulating anti-measles, anti-diphtheria,anti-polio, anti-tetanus and/or anti-varicella specific antibodies in asubject (e.g., increases the level of neutralizing titers specific forrespiratory pathogens and measles, diphtheria, polio, tetanus, and/orvaricella (e.g., thereby providing protective levels of anti-respiratorypathogen specific antibodies and anti-measles, anti-diphtheria,anti-polio, anti-tetanus and/or anti-varicella specific antibodiesbetween scheduled dates of administration of the pooled plasma and/orimmunoglobulin prepared from same that are not maintained in a subjectadministered a mixture of plasma samples obtained from 1000 or morerandom human subjects or immunoglobulin prepared from same)).

In one embodiment, the invention provides that the detection of highantibody titer within a donor plasma sample to one or more respiratorypathogens (e.g., RSV) can be used to identify (e.g., as a biomarker) aplasma donor as a high/strong responder to antigen challenge (e.g., thatpossesses high antibody titers (e.g., to a plurality of respiratorypathogens)) via generation of elevated levels of antibodies, versusdonors that are not strong responders/do not generate elevated levels ofantibodies (e.g., that possess medium to low antibody titers) (e.g., SeeExample 1). Thus, in one embodiment, the invention provides a method ofidentifying a plasma donor as a high/strong responder to antigenchallenge comprising obtaining a plasma sample from the subject,characterizing the pathogen-specific antibody titer within the plasmafor one or more respiratory pathogens selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus andcoronavirus; and identifying the subject as a high/strong responder toantigen challenge if the plasma contains elevated levels (e.g., at least1.5 fold (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 fold or more), compared to the pathogen-specific antibodytiters found in a mixture of plasma samples obtained from 1000 or morerandom human subjects, of pathogen-specific antibody titers to the oneor more respiratory pathogens. In one embodiment, the plasma compriseselevated levels of pathogen-specific neutralizing antibody titers to atleast two or more respiratory pathogens selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus andcoronavirus, that are each elevated at least 1.5 fold (e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold or more)compared to the pathogen-specific antibody titers found in a mixture ofplasma samples obtained from 1000 or more random human subjects. In afurther embodiment, the invention provides a method of using the levelof a respiratory pathogen specific neutralizing antibody titer (e.g.,RSV neutralizing antibody titer) detected in a plasma donor sample as abiomarker as an indication of the level of respiratory pathogen specificneutralizing antibody titers within the sample to one or more ofinfluenza A virus, influenza B virus, parainfluenza virus type 1,parainfluenza virus type 2, metapneumovirus and coronavirus (e.g., forthe identification, selection and blending/mixing of plasma samples).

Plasma obtained from one or a plurality of donors identified as ahigh/strong responder (e.g., containing high neutralizing antibodytiters) can be used, together with plasma from one or a plurality ofdonors identified as not being high/strong responder (e.g., do not havehigh neutralizing antibody titers) in order to generate a mixture ofplasma samples possessing a desired characteristic. For example, in oneembodiment, the invention provides a method of producing a pooled plasmacomposition comprising obtaining plasma samples from human subjects;characterizing the pathogen-specific antibody titer, within a subset ofthe plasma samples, for one or more respiratory pathogens selected fromrespiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirusand coronavirus; selecting, based upon the antibody titerscharacterized, plasma samples that have elevated levels, compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects, of pathogen-specificantibody titers to one or more respiratory pathogens selected fromrespiratory syncytial virus, influenza A virus, influenza B virus,parainfluenza virus type 1, parainfluenza virus type 2, metapneumovirusand coronavirus; pooling the selected plasma samples with other plasmasamples to generate the pooled plasma composition, wherein the pooledplasma composition comprises pathogen-specific antibody titers to atleast two or more respiratory pathogens selected from respiratorysyncytial virus, influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus andcoronavirus, that are each elevated at least 1.5 fold compared to thepathogen-specific antibody titers found in a mixture of plasma samplesobtained from 1000 or more random human subjects. In a preferredembodiment, 1000 or more plasma samples categorized according to themethods described herein are pooled in order to generate the pooledplasma composition (See, e.g., Example 2).

As described in Examples 3 and 4, use of a composition of the invention(e.g., immunoglobulin prepared from plasma pooled using selectionprocesses of the invention) provide clinical efficacy and improvedoutcomes in subjects.

Immunotherapeutic compositions of the invention were tested in a cottonrat model of human immunodeficiency. Therapeutic as well as prophylacticpotential was assessed. For analysis of therapeutic potential,immunoglobulin prepared as described in Example 2 was administeredtherapeutically to immunosuppressed (cyclophosphamide treated) andnormal cotton rats challenged with RSV/A/Long (See Example 3).

Immunosuppression in cyclophosphamide-treated groups (A, B, C, and G)was verified by significant decrease in whole blood cell and lymphocytecounts compared to normal cotton rats (groups D, E, and F).Immunosuppressed animals treated with IVIG of the invention, groups Band C, showed significant reduction in lung and nose viral load at day 4post infections (p.i). and day 10 post infection. compared toRSV-infected immunosuppressed animals treated with saline (group A) (SeeExample 3). This reduction was accompanied by reduction in lunghistopathology and a decrease in the detection of viral RNA in lung,liver, and kidney samples of group B and C animals on day 10 postinfection. Treatment of normal cotton rats with IVIG of the inventionalso resulted in significant reduction of RSV load in the lungs and noseof IGIV-treated animals (groups E and F) compared to saline-treated(group D) animals on day 4 p.i. (See Example 3).

Immunotherapeutic compositions of the invention were also tested in acotton rat model of human immunodeficiency for prophylactic potential(See Example 4). Immunosuppresion was verified by significant decreasein whole blood cell and lymphocyte counts, and reduction in serum totalIgG. Immunosuppressed animals treated with IVIG of the invention showedundetectable lung viral replication at day 4 p.i., and almost completereduction in the prolonged viral replication caused by immunosuppressionmeasured on day 10 (only two out of 5 animals in a group showed minimalviral replication (See Example 4)). This reduction was accompanied byreduction in lung histopathology and decrease in the detection of viralRNA in lung, kidney, and liver samples of selected immunosuppressedanimals.

While an understanding of a mechanism is not necessary to practice thepresent invention, and while the invention is not limited to anyparticular mechanism, in one embodiment, methods of identifying andselecting high titer respiratory pathogen antibody titers (e.g.,specific for RSV) in a subject's plasma of the invention identify and/orselect subject's that in general have an immune response system thatgenerates high levels of respiratory pathogen specific antibodiescompared to other subjects that do not display high titer respiratorypathogen antibody titers (e.g., towards RSV) and do not generate highlevels of respiratory pathogen specific antibodies. The immune responsegene that encodes for the magnitude of humoral antibody responses tomicrobial antigens is under the control of the major histocompatibilitycomplex(HLA). In this context the donors who were selected based ontheir high responses to RSV are, in some embodiments, high responders toother respiratory viruses. While an understanding of a mechanism is notnecessary to practice the present invention, and while the invention isnot limited to any particular mechanism, in one embodiment, a highresponse to respiratory pathogens is due to donors being exposed byvirtue of their occupation or other demographic considerations (e.g.,such that not only is a subject repeatedly exposed to RSV infection butalso to exposure or infection with other common respiratory viruses).Thus, in some embodiments, a subject's immunological history accountsfor the fact that the subject has elevated titers to multiple commonrespiratory viruses.

In certain embodiments, plasma and/or antibody samples comprise donatedand/or purchased body fluid samples, for example individual blood orblood component samples (e.g., plasma). These samples may be purifiedand/or screened for the presence of pathogens or other impurities (e.g.,before or after pooling). Multiple donor antibody samples (e.g., donorplasma samples or other antibody-containing samples) can pooled togetherto create a pooled plasma sample/primary antibody pool (e.g., afteridentifying or screening for desired antibody titer in the antibodysamples). By combining individual antibody samples (e.g., blood or bloodcomponent (e.g., plasma) samples) which have higher than normal titersof antibodies to one or more selected antigens, epitopes, extracellularproteins, viral surface proteins, together with plasma taken from donorsnot selected for high titers, a pooled plasma sample/primary antibodypool is created that exhibits elevated titer for such antibodies. Insome embodiments, selected antigens, epitopes, extracellular proteins,viral surface proteins, etc. are administered to subjects to induce theexpression of desired antibodies (e.g., from which antibody samples canbe harvested). The resulting enhanced high titer antibody sample (e.g.,blood, serum, plasma, purified antibodies (e.g., containing higherantibody titer as compared to a control level (e.g., the antibody titerin pooled plasma samples from 1000 or more random human subjects)) isrecovered and pooled with antibody samples from other subjectsexhibiting or anticipated to exhibit elevated titer for the sameantibodies (or antibodies directed to the same antigens, extracellularproteins, viral surface proteins, etc.), or with antibody samples fromsubject that have not been screened for antibody titer or that possess alow or absent antibody titer to a specific pathogen. In someembodiments, the pooled antibody samples are purified, screened, and/orconcentrated. In one embodiment, pooling of samples (e.g., 1000 or moresamples) occurs in a manner that uses the fewest possible number ofsamples from high titer donors (e.g., identified by the compositions andmethods described herein) but that still maintains a desired,standardized and elevated antibody titer to one or more (e.g., two,three, four or more) respiratory pathogens described herein.

Certain embodiments of the invention utilize plasma from subjects thathave been administered immunogenic substances (e.g., vaccines, antigens,epitopes, extracellular proteins, viral surface proteins, etc) in orderto generate elevated levels of specific neutralizing antibodies withinthe subject. The invention is not limited by the type of antigen (e.g.,S. pneumoniae antigen) used for administration to a subject (e.g.,donor) to induce the expression of specific antibodies. In someembodiments, the antigen is a S. pneumoniae antigen or fragment orcomponent thereof. In some embodiments, the antigen is a polysaccharide(e.g., unconjugated or conjugated to a carrier or protein) or aplurality of the same. In some embodiments, the antigen (e.g., S.pneumoniae antigen) is a S vaccine comprising components capable ofinducing specific antibodies (e.g., antibodies that are specific tomultiple different serotypes of S. pneumonia). In some embodiments, avaccine is a commercially available vaccine. The invention is notlimited by the vaccine. Indeed, a variety of vaccines (e.g., S.pneumoniae vaccines) may be utilized including, but not limited to,PREVNAR, SYNFLORIX, PNEUMOVAX as well as others known in the art.Similarly, the invention is not limited by the type or route ofadministration/immunization. Indeed, any route/type of immunization maybe utilized including, but not limited to, the methods described in U.S.Patent Publication Nos. US2008026002, US2007009542; US2002094338;US2005070876; US2002010428; US2009047353; US2008066739; andUS2002038111), each of which is hereby incorporated by reference in itsentirety. In like manner, the invention is not limited by the vaccineformulation (e.g., of a S. pneumoniae vaccine). Indeed, any formulationmay be utilized including, but not limited to, those described inUS2002107265, hereby incorporated by reference in its entirety. In someembodiments, the vaccine is a multivalent vaccine in which additionalantigens are added (See, e.g. US2007161088; US2006121059, each of whichis hereby incorporated by reference in its entirety.). In someembodiments, mirobial antigens are purified prior to use in a vaccine(e.g., a conjugate vaccine) (See, e.g., US2008286838 hereby incorporatedby reference in its entirety). Methods of culture of microorganismsuseful in a process of manufacturing a pneumococcal conjugate vaccinesare described in US2010290996, hereby incorporated by reference in itsentirety. Alternatively, in some embodiments, antigens (e.g., S.pneumonia antigens) are utilized that are not conjugated to a carrierprotein (See, e.g., US2009136547, hereby incorporated by reference inits entirety). In some embodiments, immunomodulators are utilized (See,e.g., US2004156857; U.S. Pat. No. 5,985,264; and WO11041691, each ofwhich is hereby incorporated by reference in its entirety). In someembodiments, therapeutic antibodies are produced in a donor administeredan antigen (e.g., an S. pneumoniae antigen) and/or vaccine according tothe methods described in WO05070458; US2009191217; and WO10094720, eachof which is hereby incorporated by reference in its entirety. In someembodiments, antigens (e.g., vaccines (e.g., conjugate or unconjugatedvaccines)) are used to generate antibodies (e.g., present in serumand/or plasma) that are useful against infectious disease organisms(e.g., as described in, for example, US2003099672; WO0062802, each ofwhich is hereby incorporated by reference in its entirety).

In some embodiments, a polysaccharide vaccine is used (e.g., containingmultiple S. pneumoniae serotypes (e.g., containing purifiedpolysaccharides from 1, 2, 3, 4, or more or all 23 of the following S.pneumoniae serotypes: 1, 2, 3, 4, 5, 6b, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F). Although anunderstanding of a mechanism is not needed to practice the presentinvention, and while the present invention is not limited to anyparticular mechanism of action, in some embodiments, an antigen (e.g., aS. pneumoniae antigen) or vaccine that stimulates B-cells (e.g., plasmacells) to generate and secrete specific (e.g., S. pneumonia-specific)immunoglobulin (e.g., S. pneumonia-specific IgM) without the assistanceof T cells finds use in the invention.

In some embodiments, a conjugated vaccine is utilized that containscapsular polysaccharides (e.g., from a plurality of S. pneumoniaeserotypes) that is covalently bound to a carrier and/or adjuvant (e.g.,the diphtheria toxoid CRM197). Although an understanding of a mechanismis not needed to practice the present invention, and while the presentinvention is not limited to any particular mechanism of action, in someembodiments, any antigen (e.g., S. pneumoniae antigen) or vaccine thatstimulates B-cells (e.g., plasma cells) to generate and secrete specific(e.g., S. pneumonia-specific) immunoglobulin (e.g., S.pneumonia-specific IgM and/or IgG) via interaction with specific type 2helper T cells) and/or production of memory B cells (e.g., S.pneumonia-specific memory B cells) finds use in the invention.

Thus, in some embodiments, the invention provides methods of stimulatinghigh antibody levels in a donor, which includes administering to ananimal, for example a human, a pharmaceutically-acceptable compositioncomprising an immunologically effective amount of an antigen composition(e.g., an S. pneumoniae antigen composition). The composition caninclude partially or significantly purified antigens (e.g., S.pneumoniae antigens (e.g., polysaccharide, protein and/or peptideepitopes, obtained from natural or recombinant sources, which may beobtained naturally or either chemically synthesized, or alternativelyproduced in vitro from recombinant host cells expressing DNA segmentsencoding such epitopes)).

Methods to determine the efficacy of immunization (e.g., determining thelevel of S. pneumonia-specific antibody titers) are known in the art,and any known method may be utilized to assess the efficacy ofimmunization. In some embodiments, detection methods for the evaluationof the efficacy of a vaccine (e.g., a pneumococcal conjugate vaccine) isused as described in, for example, US2005260694; U.S. Pat. Nos.4,308,026; 4,185,084; or US2005208608, each of which is herebyincorporated by reference in its entirety.

In some embodiments, kits and methods are provided that identify samplesand/or pools with specific antibody titers (e.g., antibody titers thatare elevated). In one embodiment, a suitable amount of a detectionreagent (e.g., antibody specific for antibodies, an antigen, or otherreagent known in the art) is immobilized on a solid support and labeledwith a detectable agent. Antibodies can be immobilized to a variety ofsolid substrates by known methods. Suitable solid support substratesinclude materials having a membrane or coating supported by or attachedto sticks, beads, cups, flat packs, or other solid support. Other solidsubstrates include cell culture plates, ELISA plates, tubes, andpolymeric membranes. The antibodies can be labeled with a detectableagent such as a fluorochrome, a radioactive label, biotin, or anotherenzyme, such as horseradish peroxidase, alkaline phosphatase and2-galactosidase. If the detection reagent is an enzyme, a means fordetecting the detection reagent can be supplied with the kit. A suitablemeans for detecting a detectable agent employs an enzyme as a detectableagent and an enzyme substrate that changes color upon contact with theenzyme. The kit can also contain a means to evaluate the product of theassay, for example, a color chart, or numerical reference chart. Somesuitable methods for characterizing samples and pools are provided inthe references incorporated by reference herein. The present inventionis not limited by the method used to characterize samples and pools ashaving elevated titer.

In certain embodiments, compositions are provided (e.g., antibodysamples, pooled plasma samples, immunoglobulins, etc.) in whichantibodies have been purified and/or isolated from one or morecontaminants. Human immunoglobulins were first isolated on a large scaleduring the 1940's by F. J. Cohn. In some embodiments, the techniquesprovided by Cohn (Cohn et al., J. Am. Chem. Soc. 1946; 68:459-475;herein incorporated by reference in its entirety) or modifiedCohn-techniques are utilized in preparation of immunoglobulins herein.In some embodiments, various purification and isolation methods areutilized to produce substantially unmodified, unaltered, non-denaturedand/or native immunoglobulin molecules of high purity. Exemplarytechniques are provided, for example, in U.S. Pat. No. 4,482,483, hereinincorporated by reference in its entirety. In some embodiments,compositions (e.g., antibody pools) comprise >50% immunoglobulin(e.g., >60%, >70%, >80%, >90%, >95%, >99%). Various methods may beutilized for producing such compositions, including, for example,standard protein purification and isolation techniques as well asfractionation of biological fluids (e.g., plasma). Descriptions offractionation of antibodies for use in immunotherapeutics are found, forexample in U.S. Pat. No. 4,346,073 and other references provided herein,each of which is incorporated by reference in their entireties. Incertain embodiments, immunoglobulins are purified by a fractionalprecipitation method, ion-exchange chromatography, size exclusionchromatography, ultrafiltration, affinity chromatography, or anysuitable combinations thereof (See, e.g., U.S. Pat. Nos. 7,597,891;4,256,631; 4,305,870; Lullau et al., J. Biol. Chem. 1996;271:16300-16309; Corthesy, Biochem. Soc. Trans. 1997; 25:471-475; andCrottet et al., Biochem. J. 1999; 341:299-306; herein incorporated byreference in their entireties).

In some embodiments, plasma samples are pooled to produce a large volumeof antibodies/immunoglobulins (e.g., for commercial, clinical,therapeutic, and/or research use). In particular embodiments, antibodysamples (e.g., plasma samples) exhibiting a certain desiredcharacteristic or characteristics are pooled to result in a primaryantibody pool (e.g., pooled plasma samples) enhanced for, exhibiting,and/or enriched in that desired characteristic. In certain embodiments,antibody samples (e.g., plasma) obtained from multiple subjects(e.g., >2 subjects, >5, >10 subjects, >20 subjects, >100 subjects, >200subjects, >500 subjects, >1,000 subjects, >2,000 subjects, >5,000subjects, >10,000 subjects, or more) are pooled. The subjects from whichthe antibody samples (e.g., blood, plasma, etc.) may be obtained mayhave had recent exposure to a pathogen, antigen, or epitope, beenrecently vaccinated with a pathogen, antigen, or epitope, or have beenspecifically exposed to a pathogen, antigen, or epitope for the purposeof producing specific antibodies.

In some embodiments, methods are provided for pooling/combining primaryantibody pools (e.g., pooled plasma samples) to produce secondaryantibody pools or tailored antibody pools. Two or more primary antibodypools, each exhibiting a desired characteristic (e.g., antibodiesagainst RSV, antibodies against influenza, etc.), are combined at adesired ratio to produce a tailored antibody pool. In some embodiments,a tailored antibody pool exhibits the relative sum of thecharacteristics of the primary antibody pools from which it is derived(e.g., tailored pool confers immunity to specific pathogens to an extentthat is consistent with the relative amount of the primary pools fromwhich it is derived). In other embodiments, a tailored antibody poolexhibits distinct characteristics from the primary antibody pools fromwhich it is derived (e.g., tailored pool confers immunity to a specificpathogen to a greater extent than the primary pools from which it isderived used individually, provides enhanced general immunity comparedto use of individual primary pools, provides enhanced anti-inflammatorybenefit compared to use of individual primary pools).

A composition of the invention (e.g., pooled plasma and/orimmunoglobulin prepared from same) can be administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition, compositions of the invention may be administered by pulseinfusion, particularly with declining doses. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is acute orchronic.

A composition of the invention may be formulated, dosed, and/oradministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. Compositions of the invention need notbe, but optionally are formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of acomposition of the invention (when used alone or in combination with oneor more other additional therapeutic agents) may depend upon a number offactors including the type of disease to be treated, the type ofantibody, the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, interaction with other drugs being concurrentlyadministered, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, and the patient's clinical history.

An exact dosage may be determined by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety (e.g., plasma pool) or tomaintain the desired effect. Additional factors which may be taken intoaccount include the severity of the disease state; age, weight, andgender of the patient; diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks, four weeks, sixweeks, eight weeks or more, depending on half-life and clearance rate ofthe particular formulation.

A composition of the invention may be administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 5000 mg/kg (e.g. 0.5 mg/kg-1500 mg/kg)of a composition of the invention can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. As describedherein, additional drugs or agents (e.g., antibiotics, antivirals,anti-inflammatory and/or healing compounds) may be administeredconcurrently with a pooled plasma composition of the invention. Anexemplary daily dosage of such agent may range from about 1 μg/kg to 100mg/kg or more. For repeated administrations over several days or longer,depending on the condition, the treatment can generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of a composition of the invention would be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) maybe administered to a patient. Such doses may be administeredintermittently, e.g. every week or every two or three weeks. A medicalpractitioner is readily able to monitor the therapeutic administrationof a composition of the invention and can in turn determine if higher orlower doses of the composition is to be administered.

Compositions of the invention may be administered (e.g., intravenously,orally, intramuscularly, subcutaneously, etc.) to a patient in apharmaceutically acceptable carrier such as physiological saline. Suchmethods are well known to those of ordinary skill in the art.

Accordingly, in some embodiments of the present invention, a compositionof the invention can be administered to a patient alone, or incombination with other drugs or in pharmaceutical compositions where itis mixed with excipient(s) or other pharmaceutically acceptablecarriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert. Dependingon the condition being treated, pharmaceutical compositions may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in the latest edition of“Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton Pa.).Suitable routes may, for example, include oral or transmucosaladministration; as well as parenteral delivery, including intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intraperitoneal, or intranasal administration.

For injection, a composition of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiologically bufferedsaline. For tissue or cellular administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

In other embodiments, the compositions of the present invention (e.g.,pharmaceutical compositions) can be formulated using pharmaceuticallyacceptable carriers well known in the art in dosages suitable for oraladministration. Such carriers enable the pharmaceutical compositions tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral or nasal ingestion by apatient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of a composition of the invention may be that amountthat results in the inhibition of growth and/or killing of bacteria in asubject. Determination of effective amounts is well within thecapability of those skilled in the art, especially in light of thedisclosure provided herein.

In addition to the active ingredients pharmaceutical compositions maycontain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of thecompositions of the invention into preparations which can be usedpharmaceutically.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the compositions in water-soluble form.Additionally, suspensions of the compositions may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compositionsto allow for the preparation of highly concentrated solutions.

Compositions of the invention formulated in a pharmaceutical acceptablecarrier may be prepared, placed in an appropriate container, and labeledfor treatment of an indicated condition. Conditions indicated on thelabel may include treatment or prevention of a viral or bacterialinfection.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

Compositions of the present invention (e.g., pooled plasma samples) canbe combined with additional agents (e.g., antibodies, antibodyfragments, antibody-like molecules, monoclonal antibodies, or otherproteins or small molecules) to enhance the immunotherapeutic and/oranti-inflammatory affect. Such additional agents may be producedrecombinantly, synthetically, in vitro, etc. The present invention isnot limited by the types of additional agents that a pooled antibodysample or other sample is combined with. In some embodiments,recombinant or synthetic antibodies (e.g., humanized monoclonals) orantibody fragments (e.g., directed to a specific pathogen or antigen)are added. In addition, antibodies (e.g., monoclonal, polyclonal, etc.)for specified bacteria and viruses can be added to the compositions. Insome embodiments, various therapeutics (e.g., anti-inflammatory agents,chemotherapeutics), stabilizers, buffers, etc. are added to the antibodysample pools, for example, to further enhance the efficacy, stability,administerability, duration of action, range of uses, etc.

Compositions may optionally contain carriers such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and the like. Prolonged absorption ofthe immunoglobulins can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

In some embodiments, a composition of the invention is administered to asubject to provide therapeutic, preventative, prophylactic, and/or otherbenefits.

In some embodiments, an immunotherapeutic composition of the invention(e.g., with elevated of antibodies against two or more pathogens,antigens or epitopes, etc.)) is effective in treating (e.g.,therapeutically, preventatively, prophylactically, etc.), bind antigensfrom, and/or are directed to pathogentic bacteria including, but notlimited to: Bacillus anthracis, Bordetella pertussis, Borreliaburgdorferi, Brucella abortus, Brucella canis, Brucella melitensis,Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydiatrachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Francisella tularensis, Haemophilus influenza, Helicobacterpylori, Legionella pneumophila, Leptospira interrogans, Listeriamonocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides,Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi,Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcusagalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponemapallidum, Vibrio cholera, and Yersinia pestis.

In some embodiments, an immunotherapeutic composition of the invention(e.g., with elevated of antibodies against two or more pathogens,antigens or epitopes, etc.)) are effective in treating (e.g.,therapeutically, preventatively, prophylactically, etc.), bind antigensfrom, and/or are directed to pathogentic viruses including, but notlimited to: adenovirus, coxsackie virus, Epstein-barr virus, BK virus,hepatitis a virus, hepatitis b virus, hepatitis c virus, erpes simplexvirus (type 1), herpes simplex virus (type 2), cytomegalovirus, humanherpesvirus (type 8), human immunodeficiency virus (hiv), influenzavirus, measles virus, mumps virus, human papillomavirus, parainfluenzavirus, poliovirus, rabies virus, respiratory syncytial virus, rubellavirus, and varicella-zoster virus.

Diseases and conditions for which administration of the compositions ofthe invention is to be used therapeutically or prophylactically include,but are not limited to: common variable immunodeficiency, IgAdeficiency, human immunodeficiency virus (HIV) infection, bacterial andviral infections such as respiratory tract infection with influenza,respiratory tract infection with respiratory syncytial virus,respiratory tract infection with rhinovirus, respiratory tract infectionwith adenovirus: protozoan infections such as giadiasis, yeastinfections; chronic lymphocytic leukemia; multiple myeloma;macroglobulinemia; chronic bronchitis; broncliectasis; asthma; immunesuppression associated with bone marrow transplantation; immunesuppression associated with cyclophosphamide administration; immunesuppression associated with azathiaprine administration; immunesuppression associated with methotrexate administration; immunesuppression associated with chlorambucil administration; immunesuppression associated with nitrogen mustard administration; immunesuppression associated with 6-mercaptopurine administration; immunesuppression associated with thioguanine administration; severe combinedimmunodeficiency; adenosine deaminase deficiency; majorhistocompatibility class I (Bare leukocyte syndrome) and class IIdeficiencies; purine nucleoside phosphorylase deficiency; DiGeorgeSyndrome; transient hypogammaglobulinemia of infancy; X-linkedagammaglobulinemia; X-linked agammaglobulinemia with growth hormonedeficiency; transcobalamin II deficiency; immunodeficiency with thymoma;immunodeficiency with hereditary defective response to Epstein Barrvirus; immunoglobulin deficiency with increased IgM; P chain deficiency;ataxia telangiectasia; immunodeficiency with partial albinism; sequelaeof selective IgA deficiency such as those due to rheumatoid arthritis;juvenile rheumatoid arthritis; systemic lupus erythematosus;thyroiditis; pernicious anemia; dermatomyositis; Coomb's positivehemolytic anemia; idiopathic Addison's disease; cerebral vasculitis andidiopathic thrombocytopenic purpura.

The use of specific compositions and methods of the invention to treatpathogens or treat/prevent infection may vary depending on the site ofinfection. For example, immunotherapeutic compositions used for treatingand/or preventing respiratory infections might include immunoglobulinswith antibodies and/or monoclonal antibodies specific for at least twoof the following pathogens: respiratory syncytial virus, influenza Avirus, influenza B virus, influenza C virus, parainfluenza virus type 1,parainfluenza virus type 2, rhinovirus, metapneumovirus, coronavirus, S.pneumonia, H. influenza, L. pneumophila, group A Streptococcus,Streptococcus mutans, B. gingivalis, S. pyogenes (group A), S.pneumoniae, K pneumoniae, P. aeruginosa, S. aureus, M pneumoniae, or anyother respiratory or other type of pathogen known by those of ordinaryskill in the art or described herein.

Various diseases (e.g., cancer, AIDS, etc.), infections, and treatments(e.g., antivirals, antirejections medications, chemotherapies, etc.) canresult in localized or general inflammation in a subject, which can leadto discomfort, downstream health problems, morbidity, and/or death. Insome embodiments, compositions and methods of the present inventionprovide anti-inflammatory benefits when administered to a subject.Pooled immunoglobulins have been shown to provide an anti-inflammatoryaction when passively administered (See, e.g., Nimmerjahn and Ravetch,Annu. Rev. Immunol. 2008. 26:513-33.; Ramakrishna et al. Plos Pathogens.2011. 7:6:e1002071.; herein incorporated by reference in theirentireties). In some embodiments, a composition of the invention exertsenhanced anti-inflammatory effect (e.g., 10% enhancement, 20%enhancement, 50% enhancement, 2-fold enhancement 3-fold enhancement,5-fold enhancement, 10-fold enhancement, or greater) compared to theanti-inflammatory effect of a mixture of plasma samples obtained fromrandom human subjects (e.g., 1000 or more random human subjects).Although an understanding of a mechanism is not necessary to practicethe present invention and while the present invention is not limited toany particular mechanism, in one embodiment, a pooled plasma compositionof the invention displays significantly enhanced anti-inflammatoryeffect compared to a conventional IVIG because the pooled plasmacomposition of the invention comprises plasma from at least 1000 donors(e.g., compared to a conventional hyperimmune globulin prepared from alimited number of donors (e.g., in one embodiment, the larger the numberof different plasma samples pooled, the more beneficial theanti-inflammatory effect (e.g., the greater the histopathologicalbenefit (e.g., reduction of epithelial cell death)) observed)).

In certain embodiments, compositions of the invention provide treatmentand prophylaxis of wounds, burns, nosocomial infections, and oral andrespiratory infections. In some embodiments, immunotherapeuticcompositions of the invention comprise specific antibody titers againstspecific pathogens. For example, the antibody titers for specificpathogens in the compositions of the invention may be between 1 and 1000μg/ml (e.g., 1 μg/ml . . . 2 μg/ml . . . 5 μg/ml . . . 10 μg/ml . . . 20μg/ml . . . 50 μg/ml . . . 100 μg/ml . . . 200 μg/ml . . . 500 μg/ml . .. 1000 .mu.g/ml), although higher and lower titers are contemplated.

In some embodiments, the protective activity of an immunotherapeuticcomposition comprising a tailored antibody pool is enhanced by furthercomprising one or more additional agents, including, but not limited to:antibiotics, antivirals, anti-inflammatory and/or healing compounds. Forexample, biocides, surfactants, bacterial blocking receptor analogues,cytokines, growth factors, macrophage chemotactic agents,cephalosporins, aminoglycosides, fluoroquinolones, etc., can be providedat therapeutically acceptable levels in the compositions of theinvention.

In some embodiments of the present invention, compositions of theinvention are administered alone, while in other embodiments, thecompositions are preferably present in a pharmaceutical formulationcomprising at least one active ingredient/agent, as defined above,together with a solid support or alternatively, together with one ormore pharmaceutically acceptable carriers and optionally othertherapeutic agents. Each carrier must be “acceptable” in the sense thatit is compatible with the other ingredients of the formulation and notinjurious to the subject.

Compositions of the invention can be administered via any suitable routeof administration (e.g., enteral route, parenteral route, etc.). Theterm “enteral route” of administration refers to the administration viaany part of the gastrointestinal tract. Examples of enteral routesinclude oral, mucosal, buccal, and rectal route, or intragastric route.“Parenteral route” of administration refers to a route of administrationother than enteral route. Examples of parenteral routes ofadministration include intravenous, intramuscular, intradermal,intraperitoneal, intratumor, intravesical, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, transtracheal,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal, subcutaneous, or topical administration. In typicalembodiments, compositions are administered to a subject such that theyenter the bloodstream (e.g., intravenous administration). In someembodiments, compositions are administered to devices or instrumentsthat will come into contact with a subject's body (e.g., medicaldevices, bandages, etc.). The antibodies and compositions of thedisclosure can be administered using any suitable method, such as byoral ingestion (e.g., pill, tablet, syrup, liquid, elixir, etc.),nasogastric tube, gastrostomy tube, injection (e.g., intravenous),infusion, implantable infusion pump, and osmotic pump. The suitableroute and method of administration may vary depending on a number offactors such as the specific antibody or antibodies being used, the rateof absorption desired, specific formulation or dosage form used, type orseverity of the disorder being treated, the specific site of action, andconditions of the patient, and can be readily selected by a personskilled in the art

The term “therapeutically effective amount” refers to an amount that iseffective for an intended therapeutic purpose. For example, in thecontext of enhancing an immune response, a “therapeutically effectiveamount” is any amount that is effective in stimulating, evoking,increasing, improving, or augmenting any response of a mammal's immunesystem. In the context of providing anti-inflammatory action, a“therapeutically effective amount” is any amount that is sufficient tocause any desirable or beneficial reduction in inflammation orprevention of the occurrence of inflammation. The therapeuticallyeffective amount of an antibody usually ranges from about 0.001 to about5000 mg/kg, and more usually about 0.05 to about 100 mg/kg, of the bodyweight of the mammal. For example, the amount can be about 0.3 mg/kg, 1mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, or 100 mg/kg of body weightof the mammal. The precise dosage level to be administered can bereadily determined by a person skilled in the art and will depend on anumber of factors, such as the type, and severity of the disorder to betreated, the particular binding molecule employed, the route ofadministration, the time of administration, the duration of thetreatment, the particular additional therapy employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

An immunotherapeutic composition, tailored antibody pool, or othercomposition of the invention is often administered on multipleoccasions. Intervals between single doses can be, for example, on theorder of hours, days, weeks, months, or years. An exemplary treatmentregimen entails administration once per week, once every two weeks, onceevery three weeks, once every four weeks, once a month, once every 3months or once every three to 6 months. Example dosage regimens for aimmunotherapeutic composition comprising a tailored antibody poolinclude 1 mg/kg body weight or 3 mg/kg body weight via intravenousadministration, using one of the following dosing schedules: (i) everyfour weeks for six dosages, then every three months; (ii) every threeweeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weightevery three weeks. Other dosages and regimens may be determined byclinicians, researchers, or other practitioners of the invention.

It should be understood that the immunotherapeutic compositionsdescribed herein have veterinary applications as well as human healthcare utility.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1

Studies have shown that the ability of humans to respond to foreignantigens (e.g., microbial pathogens (e.g., naturally occurring or in theform of a vaccine)) is controlled by the major histocompatibilitycomplex (human leukocyte antigen “HLA” type in humans, the majorhistocompatibility complex of the mouse, H-2, is homologous to HLA inhumans). Additional studies have shown that the histocompatibilitycomplex controls the humoral antibody responses generated within asubject against microbial pathogens. Different HLA typing programs haveexisted for some time and have studied HLA type with regard to variousimmunological responses in humans (e.g., bone marrow transplant graftand/or rejection, organ transplant, autoimmunity, cancer, and strengthof immune response (e.g., humoral immune response) generated by asubject). While HLA typing can be useful in these limited contexts,cost, medical record and other concerns make it unfeasible to HLA typeindividuals in other contexts.

Experiments were conducted during development of embodiments of theinvention in order to determine if a subset of plasma donors could beidentified as strong (e.g., high) humoral immune responders in theabsence of HLA typing. For example, the identification of individualsubjects, or a population of individuals, that are strong humoral immuneresponders may itself be useful in order to identify individuals aspotential plasma donors (e.g., for the manufacture of immunoglobulin).In addition, experiments were conducted in order to determine ifindividuals could be identified that were strong responders not only toa single microbial pathogen but to a plurality of microbial pathogens(e.g., such an individual, or population of individuals, may containhigh titers due to the strength of humoral immune response in thesubject, not just to a single microbial pathogen/antigen but to aplurality of microbial pathogens/antigens (e.g., any or all of themicrobial pathogens/antigens to which the individual, or population ofindividuals, had been exposed in the course of their lifetime(s)). Thus,experiments were conducted in an effort to identify plasma donors asgenerally high responders in the absence of having to tissue type (e.g.,HLA type) the donors for a specific histocompatibility gene complex. Tothis end, plasma donor samples were studied and characterized forantibody titers for one or a plurality respiratory pathogens in order tocharacterize the donors (e.g., as a high/strong responder to antigenchallenge via generation of elevated levels of antibodies versus donorsthat are not strong responders/do not generate elevated levels ofantibodies (e.g., via determining antibody titers to one or morerespiratory pathogens in the subjects)).

Respiratory pathogens were chosen because individuals are ubiquitouslyexposed to a plurality of respiratory pathogens. That is, almost alladult and pediatric human populations have been exposed to a pluralityof respiratory pathogens and would have therefore generated at somepoint in their lifetime a humoral antibody response that is measurable.Experiments were conducted in order to determine if high/strongresponders could be identified using antibody titers to one or aplurality of respiratory pathogens. In one non-limiting exampledescribed below, experiments were conducted in order to determine ifantibody titer to respiratory syncytial virus (RSV) in a donor plasmasample could be used to predict the antibody titer to other respiratorypathogens in the donor plasma sample. For example, experiments wereperformed in order to determine if high antibody titer to a respiratorypathogen (e.g., RSV) could be used as a biomarker to identify a donor asan overall high/strong responder to antigen challenge (e.g., to aplurality of respiratory or other pathogens) via generation of elevatedlevels of antibodies, versus donors that are not strong responders/donot generate elevated levels of antibodies.

Twenty random plasma donor samples were obtained and the antibody titersto a plurality of respiratory viruses determined. Specifically, theplasma donor samples were studied and characterized for antibody titersfor a variety of respiratory pathogens including respiratory syncytialvirus (RSV), influenza A (Flu A), influenza B (Flu B), parainfluenzatype 1 (PIV1), type 2 (PIV2) and type 3 (PIV3), metapneumovirus (HMPV)and/or coronavirus (strains OC43, and 229E). In order to determineantibody titers, each plasma sample was purified; the purified Igfraction was analyzed either by neutralization assay (e.g., for RSV) orenzyme-linked immunosorbent assay (ELISA) assay (e.g., influenza A andB, parainfluenza, metapneumovirus, and coronavirus). As described below,titers to each respiratory virus were obtained from each of threeseparate runs/analysis (labelled as RUN 1, RUN 2, and RUN 3), and theRSV neutralization titer for each sample was compared to the non-RSVrespiratory virus titers.

In order to quantitatively measure the titer level of neutralizinganti-RSV antibody present (e.g., in samples of human serum, plasma orproducts derived from these (also referred to as an analyte)), an RSVmicroneutralization assay was designed. Briefly, one or more dilutionsof analyte (50 μL) were incubated with an infectious stock of RSV(RSV-A2, 50-100 pfus, 50 μL) in 96-well tissue culture plates for 30minutes at room temperature. The assay diluent was the same as thegrowth media, Eagle's Minimum Essential Medium with glutamine, 2% FBSand penicillin-streptomycin (hereafter EMEM). Depending on the relativetiter of the neutralizing antibody in the analyte sample, the antibodywill neutralize some or all of the RSV. An equal volume (100 μL) ofapproximately 1.5×10⁵ HEp-2 cells (ATCC CCL-23) in EMEM were added tothe analyte/virus mixture and the samples incubated at 36-38° C.,4.5-5.5% CO₂ for 3 days, at which time only the non-neutralized viruswill propagate in the HEp-2 cells. Therefore, the amount of virus thatreplicates was inversely proportional to the amount of neutralizingantibody present in the analyte. After the incubation the virus wasfixed to the tissue culture wells with 80% acetone in PBS. The plateswere then developed by an enzyme immune-type assay (EIA). The EIAutilized a mouse-monoclonal antibody to the RSV F-protein, followed by ahorse-radish peroxidase enzyme-linked conjugate antibody that detectedthe mouse-monoclonal to the RSV F-protein. Finally, a chromogenicsubstrate was added and the EIA analyzed using standardspectrophotometric techniques. The relative absorbance values obtainedwere directly proportional to the amount of RSV virus in the wells, andthus inversely proportional to the amount of neutralizing antibody inthe analyte.

For plasma and serum titers, samples and controls were diluted 2-fold,in duplicate from 1:100 to 1:25,600 across a 96-well tissue cultureplate and incubated with virus and HEp-2 cells as described above. Inaddition, each plate contained wells with no virus (100% inhibition) andvirus without antibody (0% inhibition). These absorbance values wereused to calculate the 50% inhibition point.

Titers were defined as the minimal dilution of analyte that crossed the50% inhibition point. All titers were corrected to a standard MQC titervalue.

In order to identify and categorize samples as high, medium or low forRSV neutralization titer, assay controls were generated using threeseparate and distinct samples as well as a reference product control.The assay controls/standards were developed based on analysis ofthousands of donors analyzed during experiments conducted duringdevelopment of the invention. A low quantity control (LQC) wasidentified and used that contained RSV neutralization titers below theaverage range of titers observed, a medium QC was identified and usedthat contained RSV neutralization titers falling in a middle rangeobserved, and a high QC was identified and used that contained RSVneutralization titers at the high end of the assays (accordingly, LQC,MQC and HQC controls were identified and used based upon observations ofthousands of samples). For the assay to be considered valid, dataobtained from the controls had to meet pre-determined titer and/orabsorbance criteria. The relative RSV titer ranges for the low, mediumand high assay controls were as follows: 1800-3299; 3300-7799; and 7800or above, respectively.

For plasma and serum screening, samples and serum controls were heatinactivated and diluted 1:200 in EMEM. The 1:200 dilution was added toduplicate wells and RSV neutralization assay performed as describedabove. Absorbance ranges were determined for the LQC, MQC and HQCcontrols. The average of the duplicate absorbance values for each samplewas recorded. Again, the absorbance was inversely proportional to theamount of neutralizing antibody in the sample. Therefore, the lower theabsorbance the more neutralizing antibody present.

The LQC, MQC and HQC were run in a bracketed fashion for each plate. Thetiters were required to meet predefined criteria for the assay to bevalid. In the instance that a plasma or serum titer was <100, it may bediluted as low as 12.5 fold and 2-fold dilutions tested from 12.5 to3200. Thus, when a clinical sample was prescreened and the sample titerso low that a dilution of 1:100 would be too large a dilution to allowfor any signal detection, dilution was begun at 1:12.5 proceeded by twofold dilutions from that point. This prescreening of clinical sampleslead to the result that if the donor titer was so low as to require thistype of dilution, they were categorized as low titer. All plates wererun with control antibody bracketing the plates.

Enzyme immunoassay (EIA) was performed to detect virus-specific serumIgG for nine respiratory viruses: influenza A and B, RSV, parainfluenza(PIV) virus serotypes 1, 2 and 3, human metapneumovirus (hMPV), andcoronavirus 229E (CoV 229E) and coronavirus OC43 per published methods(See, e.g., Falsey et al., J Am Geriatr Soc. 1992; 40:115-119; Falsey etal., J Am Geriatr Soc. 1995; 43:30-36; Falsey et al., J Am Geriatr Soc.1997; 45:706-711; Falsey et al., J Infect Dis. 2003; 187:785-790).Briefly, antigens were produced from virally infected whole cell lysatesfor all viruses except RSV. Purified viral surface glycoproteins wereused as antigen for RSV EIA according to published methods (See, e.g.,Falsey et al., J Am Geriatr Soc. 1992; 40:115-119). Serial two-folddilutions of each sample were tested in duplicate.

Data analysis was performed via a paired-data approach. Data pairs werecreated by matching the donor ID within each ELISA assay run.

Specifically, to evaluate the correction between the titers to RSV andthe titers to non-RSV respiratory virus at the donor level, antibodydata were paired by matching the donor ID within the same ELISA AssayRun (referenced as RUN No. 1, Run No. 2 and Run No. 3). Titers to RSVfrom a donor were paired with the titers to another non-RSV virus of thesame donor. Hence, a total of 20 pairs were created within a Run and atotal of 60 pairs were created within a comparison. Linear correlationwas assessed between the Titers to RSV and the titers to another non-RSVvirus using Pearson correlation coefficient on linear scale and on log 2scale. All analysis was performed using SAS version 9.3.

As shown in Table 1, a positive linear correlation was found between theobserved RSV titers and the observed titers to other non-RSV respiratorypathogens. This observation indicated that plasma samples identified ashaving a high RSV titer value also possessed higher/greater titer toother non-RSV pathogens. All correlation coefficients were statisticallysignificant (p<0.05) with the estimated correlation coefficients rangingfrom 0.29 (OC43) to 0.67 (PIV 3) on the log 2 scale. A scatter plotshowing the RSV neutralization titer compared to the antibody titers forFlu A, Flu B, PIV 1 and PIV 3 in Linear Scale is shown in FIG. 1. Ascatter plot showing the RSV neutralization titer compared to theantibody titers for Flu A, Flu B, PIV 1 and PIV 3 in Log 2 Scale isshown in FIG. 2.

TABLE 1 Linear Correlation Coefficient Between Titers to RSV and Titersto Non-RSV Virus Pearson Linear Correlation Coefficients of Titers toRSV and Titers to Scale Flu A Flu B HMPV PIV1 PIV2 PIV3 OC43 229E Log20.49^(#) 0.54^(#) 0.35^({circumflex over ( )}) 0.56^(#)0.41^({circumflex over ( )}) 0.67^(#) 0.29* 0.41^(#) Linear 0.49^(#)0.59^(#) 0.28* 0.50^(#) 0.24^(~) 0.59^(#) 0.34^({circumflex over ( )})0.40^({circumflex over ( )}) ^(#)P-value ≤ 0.001;^({circumflex over ( )})P-value ≤ 0.01; *P-value ≤ 0.05; ^(~)P-value >0.05

Additional experiments were performed, during development of embodimentsof the invention, in order to determine if the positive linearcorrelation found between the observed RSV titers and titers to othernon-RSV respiratory pathogens could be attributed to an overall increasein all titers.

For this experiment, measles neutralization titer was characterized anddetermined if it could be used to identify samples as possessing adesired characteristic (e.g., elevated levels antibody titers to othernon-measles pathogen). Two separate samples were characterized. Batch 1displayed a measles neutralization titer of 2.34 and an RSVneutralization titer of 17,275. Batch 2 displayed a significantly lowermeasles neutralization titer of 0.73, and an RSV neutralization titer of20,375. Thus, even though the measles titer in one case was roughly only30% the value of another batch, both batches correlated with/selectedfor high titer RSV. Accordingly, measles virus was determined not to beuseful as a discriminatory marker for selecting plasma samples that arehigh titer for other, non-measles viruses.

Thus, for each of the 20 random samples analyzed, there was asignificant correlation between the neutralizing antibody titer specificfor RSV and the antibody titer specific for one or more otherrespiratory pathogens—a high antibody titer to RSV directly andsignificantly correlated with high antibody titer(s) specific for otherrespiratory pathogens.

Example 2

Further experiments were conducted in order to determine if plasmasamples identified as high/strong responder (high titer) in Example 1could be combined with other, non-high titer samples in order togenerate a pool of 1000 or more samples, yet that contained a desired,elevated antibody titer to RSV and a desired, elevated antibody titer toone or more other respiratory pathogens.

Plasma donor samples were initially screened (prescreened as describedin Example 1) using optical density for RSV neutralization antibodytiter. Briefly, donor plasma diluted 1:400 was mixed with RSV and thenoverlaid on Hep 2 cells. Neutralizing activity in the plasma wasmeasured via the absence of infection of the hepatocytes. Afterincubation to allow for viral expression the plate was fixed and thenstained with an anti-RSV monoclonal antibody followed by counterantibody conjugated to horse radish peroxidase. The lower the ODobserved the higher the neutralizing power of the plasma (fewer virusesleft to infect the cells).

The top 20% of the donor samples identified as having the lowest OD's inthe prescreen were further characterized. That is, once clinicalsamples, prescreened by determining a single titration point vianeutralization assay, were identified that fell within the top 20% ofdonor samples with regard to neutralization titer, additional plasma wascollected from the donors so identified for further analysis. Theadditional donor plasma samples were purified, the immunoglobulinfraction purified/prepared, and the fraction analyzed by neutralizationassay using a full titration curve in order to obtain RSV neutralizationtiter. The donor plasma subjected to further testing was seriallydiluted (e.g., 1:100 1:200 1; 400 etc.) in a full titration assay andthe titer assigned to the sample (that is, to the plasma donor) as thatdilution that gave 50% inhibition of virus growth (50% inhibition isthat point which is 50% of the two extremes (saline plus virus is 100growth and no virus added is 0 growth)).

Immunoglobulin obtained from the additional plasma samples thatregistered an RSV neutralization titer of 1800 and above resulted in thedonor being scored as a high/strong responder. The specific RSVneutralization titer for each plasma sample from each donor was recordedand used in a subsequent blending process. Through experiments conductedduring development of embodiments of the invention, it was determinedthat only about 25-50% of those who fell into the top 20% via theinitial/pre-screen were classified using the second RSV neutralizationassay as high/strong donors based on the titer of 1800 or above (only5-10% of the total starting population screened).

Plasma was collected on an ongoing basis from donors classified ashigh/strong responders and tested periodically (monthly). During thisprocess, if a high/strong responder's RSV neutralizing titer wasdetermined to have dropped below 1700, the donor was no longerclassified as a high/strong responder. Donors identified as high/strongresponders with a RSV neutralizing titer of 1800 or higher werecategorized as high titer (high titer selected donors). Plasma from theremaining 50% of plasma donors identified as being in the top 20% of alldonors but that did not have an RSV neutralization titer of 1800 orabove were categorized as medium titer. A third group of plasma donorswere categorized as non-tested/non-selected source donors.

Plasma from high titer selected donors, non-high titer selected donorsand non-selected source donors were combined. The RSV neutralizationtiter of the mixed/blended plasma samples was calculated arithmeticallyas follows: Multiply titer of the plasma by its volume to get totaltiter and then divide by the total volume of all the plasma samples. Forexample, if one liter of plasma titer of 100 was added to one liter ofplasma titer 200 the total titer is 100×1+200 xl which is equal 300divided by 2 liter for a final titer of 150. It was tested whether itwould be possible to generate a mixed plasma from 1000 different donorswith a total volume of 2500 liters and an RSV neutralization titer of1800 or greater (and/or a RSV titer of 1800 or greater and an elevatedtiter to one or more other respiratory viruses). Because the target RSVneutralization titer was 1800, if the mix arithmetically generated ahigher value, then normal source plasma was mixed in. The titer fornon-tested/non-selected source donor plasma was set at zero. Therefore,when normal source plasma was added there was no increase in titer valueonly an increase in the volume (denominator) which, as described above,resulted in a lower final RSV neutralization titer. Blending proceededeither adding high titer material or normal source plasma until the atarget titer was reached. Attempts were made in order to generate apooled plasma composition containing about 2500 liters (L) from at least1000 human plasma donors with a final RSV titer of 1800, but utilizingsignificantly less than all high titer selected donors in the pool(e.g., due to the limited availability of high titer donors).Surprisingly, through experiments conducted during development ofembodiments of the invention, it was determined that plasma samplescould be categorized and blended in order to generate 2500 liters ofpooled plasma from at least 1000 donors with a final RSV neutralizationtiter of 1800, wherein less than half of the donors used for poolingwere high titer selected donors (e.g., identified by the two stepscreening processes described above (See Table 2, below) and where thepooled plasma composition contained significant levels of antibodytiters specific for measles, diphtheria and/or polio.

TABLE 2 Characteristics of several pooled plasma compositions of theinvention. Lot # 1 2 3 4 Total Volume (L) 2196 2192 2195 2199 No. plasmadonors 1022 1021 1035 1033 % High-titer 36 39 45 44 selected donors %Non-high titer 64 61 55 56 selected donors RSV titer of 1805 1805 18061804 pooled plasma composition

Accordingly, the invention provides a blending/pooling process thatprovides a pooled plasma composition or immunoglobulin prepared fromsame that contains a standardized and reproducible level of respiratorypathogen (e.g., RSV) specific antibodies thereby providing a heretoforeunavailable, consistent and reproducible immunoglobulin product (e.g.,for use as IVIG). Experiments confirmed that a pooled plasma compositionor immunoglobulin prepared from same of the invention (e.g., 2500 litersof pooled plasma from 1000 donors with a final RSV neutralization titerof 1800) could be consistently generated from different groups of 1000donors. Further experiments confirmed that a pooled plasma compositionof the invention (e.g., 2500 liters of pooled plasma from 1000 donorswith a final RSV neutralization titer of 1800) contained antibody levelsto tetanus, measles and polio that prevent, or protect from, infectionwith same, and also contained elevated antibody titer(s) specific forthe respiratory pathogens described in Example 1.

Upon completion of mixing of the samples, the arithmetic calculation wasrepeated (with the separate volumes for each donor with theircorresponding titers) in order to verify that the original blendingequation was correct. Thus, although each lot prepared according to thismethod possesses a different ratio of high titer selected donors andnon-high titer selected donors, the screening and blending methodsidentified and described herein provide a blended plasma product thatpossesses a standardized, elevated anti-RSV-neutralization titer as wellas elevated levels of respiratory pathogen-specific antibody titers.Once generated, the various mixture of 1000 donor plasma samples wereutilized to manufacture IVIG.

All IVIG manufacturing activities were conducted following GoodManufacturing Practices (GMP) so as to minimize contamination and ensurethe purity, identity, and potency of the drug substance. Themanufacturing process followed the modified Cohn-Oncley cold alcoholfractionation process which isolates the immunoglobulin fraction as asolution (See, e.g., Cohn e al., J Am Chem Soc, 62, 459-475 (1946); andTeschner et al., Vox Sang. 2007 January; 92(1):42-55, each of which isherein incorporated by reference in its entirety). The Cohn-Oncleymethod is a multi-step process of isolating immunoglobulins from plasmausing different alcohol concentrations under specific conditions oftemperature, pH, protein concentration and ionic strength at each step.Following fractionation, the intermediate drug substance is subject tovirus inactivation/removal steps, further purification, and formulationinto the bulk drug substance.

The modified Cohn-Oncley cold alcohol fractionation began with poolingof a sufficient number of plasma units to obtain 1800-2000 L of plasma(˜2500 units, representing at least 1000 separate donors). It wasverified that the plasma units met all regulatory requirements andPlasma Protein Therapeutics Association standards for human sourceplasma.

Cryoprecipitate was removed by centrifugation at <5° C. before thefractionation began. The cryopoor plasma was brought to pH 7.3±0.1 and8±3% SDA-3A Ethanol by weight and held at −2±1° C. while Fraction Iprecipitated. Fraction I was removed by centrifugation and thesupernatant further processed.

Fraction I supernatant was brought to pH 7.9±0.1 and 25±3% SDA-3AEthanol by weight and held at −5±1° C. while Fraction II+IIIprecipitated. Fraction II+III were removed by centrifugation,resuspended in sodium phosphate buffer at pH 7.2±0.1. SDA-3A Ethanol wasadded to 20±3% and Fraction II+IIIw was formed while the mixture washeld at −5±1° C.

Fraction II+IIIw was resuspended and Fraction III was removed at pH5.2±0.05, 17±3% SDA-3A Ethanol and temperature of −5±1° C. Fraction IIIwas separated by centrifugation and the supernatant further processed.

Acid washed Celite was added to the Fraction III supernatant and thenremoved by depth filtration. The filtered supernatant was brought to aconcentration of <70 g protein/L, pH4.2 0.25 and conductivity ≤5 mSprior to viral inactivation with 0.3±0.1% TnBP and 1.0±0.2% Triton-X 100at 28±2° C. The TnBP and Triton-X-100 were removed by C-18chromotography. The C-18 eluate was further purified using a Q-Sepharosecolumn.

Additional virus removal was achieved using 35 nm nanofiltration of theQ-Sepharose eluate. After nanofiltration the product was formulatedusing ultrafiltration/diafiltration.

Three separate, high titer RSV neutralizing antibody batches of IVIG(RSV-IVIG) were manufactured (from plasma samples pooled from 1000 ormore subject and that contained a standardized, elevated antibody titerto RSV utilizing the prescreening and screening compositions and methodsof the invention). The three RSV-IVIG were compared to 7 different lotsof commercially available, conventional IVIG (4 differentmanufactures/brands). ELISA assays were performed in order to quantitateantibody titer to RSV, PIV1, PIV2, OC43, 229E, FluA, and Flu B. TheELISA assays were run on three separate dates.

ELISA testing of IVIG was performed blinded to the type of sample. Allsamples were diluted with sample dilution buffer (PBS with 0.3% Tween 20and 0.1 M EDTA) to a standard concentration of 50 mg of IgG per ml. Eachviral antigen was diluted at previously determined concentration inbicarbonate buffer and coated separately on enzyme immunoassaymicrotiter plates and stored overnight in humidified chambers at 4° C.The following day, plates were washed and eight serial 2-fold dilutionsin duplicate of unknown product were incubated on the antigen plates atroom temperature in humidified chambers for 3 hours. The initialdilution of IVIG solution placed on antigen plates was 1:1600. Plateswere then washed and bound IgG was detected with alkaline phosphataseconjugated goat anti-human IgG followed by substrate. A standard serumwas included on each plate and the IgG titer for a specific virus wasdefined as the highest dilution with an optical density (OD) of 0.20.

Statistical Analysis. Titer data was tabulated with descriptivestatistics of N (sample size, mean, geometric mean, standard deviation,minimum, median, and maximum). Difference between the RSV-IVIG andcommercial IVIG (that is, Group 1 vs Group 2) were presented as theratio of geometric means (RGM) and 95% Confidence intervals for the RGMwas also provided. P-value for testing of the null hypothesis that theRGM equaled to 1 was produced based on 2-sample t-test at significancelevel of 0.05.

FIG. 3 demonstrates that the geometric mean antibody titer of theRSV-IVIG was significantly greater for other respiratory viruses ascompared to the geometric mean antibody titer of the commercial lots ofIVIG. In particular, there was a 1.5 to 1.8 fold greater antibody titerto the other respiratory viruses in the RSV-IVIG (group 1) as comparedto the commercial IVIG (group 2).

The properties of the IVIG from 1000 or more samples containing elevatedlevels of neutralizing antibody titers to one or more respiratorypathogens generated using the compositions and methods of the inventionis a significant advancement and improvement over other IVIG availablein the art. In particular, the IVIG compositions of the invention do notdisplay or possess a neutralizing antibody titer for only a singlepathogen (e.g., dominance for only one type of respiratory pathogens),but rather, through the methods of identifying donors and the blendingprocesses developed and described herein, IVIG is provided that containssignificantly elevated neutralizing titers to a plurality of respiratorypathogens and other pathogens (e.g., polio, diphtheria, etc.), comparedto the titers in 1000 randomly mixed plasma samples. The discovery ofthe use of neutralizing antibody titer to RSV (or other respiratorypathogen) as a biomarker to identify plasma donors that are high-titerselected donors (high/strong responders in general to respiratorypathogens (e.g., influenza A virus, influenza B virus, parainfluenzavirus type 1, parainfluenza virus type 2, metapneumovirus, coronavirus,S. pneumonia, H. influenza, L. pneumophila, and group A Streptococcus))makes possible the ability to identify donors and plasma that can beblended with non-high titer selected donors and non-selected donorplasma to provide a beneficial pooled plasma product. Thus, while anunderstanding of a mechanism is not needed to practice the presentinvention, and while the present invention is not limited to anyparticular mechanism of action, in some embodiments, the inventionprovides a heretofore unavailable pooled plasma composition (e.g.,prepared according to the above described methods)) that contains asignificant amount (e.g., greater than 50%) of non-high titer selecteddonor plasma (non-high titer RSV plasma) that provide therapeuticbenefit not achievable with standard hyperimmune immune globulin (e.g.,prepared from a limited number (e.g., 100-300) of plasma donors). In afurther embodiment, due to the elevated levels of neutralizing antibodytiters to one or a plurality of RSV, influenza A virus, influenza Bvirus, parainfluenza virus type 1, parainfluenza virus type 2,metapneumovirus, coronavirus, S. pneumonia, H. influenza, L.pneumophila, and group A Streptococcus, such pooled plasma compositionsprovide a significantly improved therapeutic benefit to a subjectadministered the composition. For example, a pooled composition of theinvention, compared to pooled plasma samples obtained from 1000 or morerandom human subjects, provides viral neutralization properties againstone or a plurality of respiratory pathogens or other pathogens that isnot provided for by randomly pooled samples (e.g., provides a subjectprophylactic and/or therapeutic levels of antibodies to polio,diphtheria and/or measles). In this way, a subject administered acomposition of the invention is able to fight off, or be treated for,infections that are not treatable with a composition of pooled plasmasamples obtained from 1000 or more random human subjects or that are nottreatable with a conventional hyperimmune immune globulin. For example,a pooled plasma composition according to the invention (e.g., from 1000or more samples wherein the pooled plasma composition comprises aneutralizing RSV antibody titer of 1800 or above and elevated levels ofantibodies to one or more respiratory pathogens) when administered to asubject provides the subject the ability to fight off, or be treatedfor, infections that are not treatable with a composition of pooledplasma samples obtained from 1000 or more random human subjects and/orthat are not treatable with a conventional hyperimmune immune globulinprepared from limited numbers of donors (e.g., such hyperimmune immuneglobulin requires a subject to be vaccinated against diphtheria, polioand/or measles in addition to receiving the hyperimmune immuneglobulin).

For example, IVIG prepared according to the methods described above wascompared with a conventional RSV-specific immune globulin available inthe art, RESPIGAM (MEDIMMUNE, Inc., hyperimmune immune globulin preparedfrom several hundred (100-300) healthy human plasma donors that have ahigher than normal concentration of antibodies specific for RSV). WhileIVIG of the invention displayed a similar level of RSV neutralizingactivity to that of RESPIGAM, neutralizing antibody titers to otherrespiratory viruses were significantly higher in the IVIG of theinvention (Table 3, IVIG-1000 donors) compared to the levels found inRESPIGAM (See Table 3).

TABLE 3 Neutralization titers for respiratory pathogens. OC43 229E PIV1(corona- (corona- RSV (paraflu) PIV2 virus) virus) IVIG-1000 204,25336,107 72,214 102,126 144,289 donors RESPIGAM 204,253 12,765 25,53136,107 25,531

IVIG prepared according to the methods described above (RSV-IVIG) wasadministered in a randomized, double blind dose range study inimmunocompromised patients infected with RSV (a phase II multicenterstudy carried out at centers in the U.S., Canada, Australia and NewZealand). Immunocompromised patients were either bone marrow transplantor solid organ transplant patients that were concurrently onimmunosuppressive treatment between the ages of 2-65 (mean age of 38years). Upper respiratory RSV infection was confirmed in each patientusing RT-PCR at time of enrollment. Patients fell into one of threearms. The first arm received 1500 mg/kg RSV-IVIG on day one followed by750 mg/kg RSV-IVIG on day two. The second arm received 750 mg/kgRSV-IVIG on day one followed by 750 mg/kg RSV-IVIG on day two. The thirdarm received saline placebo on both days. On day 18, patients in arm 1displayed a mean fold increase from baseline of anti-RSV neutralizationtiters of 9.24 (p value relative to placebo=0.0043; with 85.7% ofpatients displaying a greater than 4 fold increase). On day 18, patientsin arm 2 displayed a mean fold increase from baseline of anti-RSVneutralization titers of 4.85 (p value relative to placebo=0.0268; with42.9.% of patients displaying a greater than 4 fold increase). Antibodytiters to Streptococcus pneumonia before and after administration ofIVIG to the patients was also assessed. In general, there was nosignificant increase or decrease in the Streptococcus pneumonia specificantibody titers in the patients post administration of IVIG.

Example 3 IVIG Therapeutic Potential

Immunotherapeutic compositions of the invention were tested in a cottonrat model of human immunodeficiency. Therapeutic as well as prophylacticpotential was assessed. For analysis of therapeutic potential, RSV-IVIGprepared as described in Examples 1 and 2 above was administeredtherapeutically to immunosuppressed (cyclophosphamide treated) andnormal cotton rats challenged with RSV/A/Long.

Animals: Fifty-three (53) inbred male and female Sigmodon hispiduscotton rats between 6 to 8 weeks of age (Source: Sigmovir Biosystems,Inc., Rockville Md.) were maintained and handled under veterinarysupervision in accordance with the National Institutes of Healthguidelines and Sigmovir Institutional Animal Care UtilizationCommittee's approved animal study protocol (IACUC Protocol #2). Cottonrats were housed in clear polycarbonate cages individually and providedwith standard rodent chow (Harlan #7004) and tap water ad lib.

Challenge Virus: The prototype Long strain of RSV (ATCC, Manassas, Va.)was propagated in HEp-2 cells after serial plaque-purification to reducedefective-interfering particles. A pool of virus designated ashRSV/A/Long Lot #041513 containing approximately 5.0×107 pfu/mL insucrose stabilizing media was used for in vivo experiments. This stockof virus was stored under −80° C. conditions and has been characterizedin vivo using the cotton rat model and validated for upper and lowerrespiratory tract replication.

Test Article(s): Immunoglobulin (at 100 mg/mL) for intravenousadministration (IVIG) was obtained as described in Examples 1 and 2above and stored at 4° C. until the start of the in vivo use. Briefly,plasma samples pooled from 1000 or more subjects with a total volume of2500 liters was generated that contained a standardized, elevatedantibody titer to RSV (neutralization titer of 1800) utilizing theprescreening and screening compositions and methods of the invention,from which immunoglobulin was prepared as described in Example 2.

Methods.

Identification of animals was performed using ear tags. Bleeding wasperformed using retro-orbital sinus bleed. Collection in EDTA tubes forwhole blood analysis (day −3), collection of serum (prebleed, day 4p.i., day 10 p.i.). Route of infection was intranasal (i.n.)inoculation. Route of IVIG treatment was intra-peritoneal injection(i.p.). Euthanasia was performed using CO2 asphyxiation. Lung tissue,liver tissue, and kidney tissue were harvested post euthanasia for RSVplaque assays, quantitative PCR (qPCR) and histopathology. Table 4provides the Experimental Study Design for these experiments.

# of Treatment Volume of i.p. Group Animals Treatment Route Typetreatment* Sac Immunosuppressed: A 10 Saline i.p. dl, 4, 7 Therap. 1.5ml/1.5 ml/1.5 ml d 4, d 10 B 10 IGIV 1,500 mg/kg i.p. dl, 4, 7 Therap.1.5 ml/1.5 ml/1.5 ml d 4, d 10 (High/High/High Dose) C 10 IGIV 1,500;750 mg/kg i.p. dl, 4, 7 Therap. 1.5 ml/0.75 ml/0.75 ml d 4, d 10(High/Low/Low Dose Normal: D 10 Saline i.p dl Therap. 1.5 ml d 4, d 10 E5 IGIV 1,500 mg/kg i.p dl Therap. 1.5 ml d4 F 5 IGIV 750 mg/kg i.p dlTherap. 0.75 ml  d4 G** 3 TBD *Volume of saline or 10% IgG stocksolution administered i.p. per 100 g weight **Group G: extra 3 animalsto undergo cyclophosphamide treatment. These animals are to be used forpotential mortalities during the first three weeks of the study. Groupassignment is to be determined (TBD) on the day of RSV infection (d 0).

A schematic of the cotton rat model utilized to study the therapeuticpotential of RSV-IVIG prepared as described in Example 2 is shown inFIG. 4.

Whole Blood Assay. Automated whole blood analysis was carried out onblood samples collected in EDTA-containing tubes. Total number of whiteblood cells and lymphocytes was analyzed.

RSV plaque assay. Lung homogenates were clarified by centrifugation anddiluted 1:10 and 1:100 in EMEM. Confluent HEp-2 monolayers in 24-wellplates were infected in duplicates with 50 μl of sample per wellstarting with undiluted (neat) samples followed by diluted homogenates.After one hour incubation at 37° C. in a 5% CO₂ incubator, wells wereoverlayed with 0.75% methylcellulose medium and plates restored into the37° C. incubator. After 4 days of incubation the overlay was removed andthe cells were fixed with 0.1% crystal violet stain for one hour, thenrinsed and air-dried. Plaques were counted and viral titers wereexpressed as plaque forming units per gram of tissue. Viral titer for agroup was calculated as the geometric mean+standard error for allanimals in that group at a given time. Student-t test was applied todetermine significance of change in viral replication betweenvehicle-treated and test groups, with p<0.05 indicating astatistically-significant difference.

Real-time PCR. Total RNA was extracted from homogenized lung, kidney orliver tissue using the RNeasy purification kit (QIAGEN). One μg of totalRNA was used to prepare cDNA using QuantiTect Reverse Transcription Kit(Qiagen). For the real-time PCR reactions the QuantiFast SYBR Green PCRKit (Qiagen) was used in a final volume of 25 μl, with final primerconcentrations of 0.5 μM. Reactions were set up in 96-well trays.Amplifications were performed on a Bio-Rad iCycler for 1 cycle of 95° C.for 3 min, followed by 40 cycles of 95° C. for 10 sec, 60° C. for 10sec, and 72° C. for 15 sec. The baseline cycles and cycle threshold (Ct)were calculated by the iQ5 software in the PCR Base Line Subtracted

Curve Fit mode. Relative quantification of DNA was applied to allsamples. The standard curves were developed using serially-diluted cDNAsample most enriched in the transcript of interest (e.g., lungs from day4 post-primary RSV infection). The Ct values were plotted against log₁₀cDNA dilution factor. These curves were used to convert the Ct valuesobtained for different samples to relative expression units. Theserelative expression units were then normalized to the level of β-actinmRNA (“housekeeping gene”) expressed in the corresponding sample. Foranimal studies, mRNA levels were expressed as the geometric mean±SEM forall animals in a group at a given time.

Results.

Whole Blood Assay. Total White Blood Cell counts and total Lymphocytecounts were reduced in all cyclophosphamide treated animals (groups A,B, C, and G) compared to normal, unmanipulated cotton rats (groups D, E,and F). This difference was seen in samples collected 18 days after thebeginning of cyclophosphamide treatment (Day −3 with respect to RSVchallenge (See FIG. 6).

Pulmonary Histopathology. Pulmonary histopathology was evaluated inRSV-infected immunosuppressed animals from groups A, B, and C and innormal saline-treated and RSV-infected animals (group D) on day 10 p.i.Lungs of RSV infected immunosuppressed animals displayed increasedepithelial damage compared to the lungs of RSV-infected normal cottonrats (group A compared to group D) (FIG. 7). Treatment of animals withIGIV (groups B and C) resulted in reduction of epithelial damage. Nosignificant differences were noted for the two different regimes of IVIGtreatment used in groups B and C.

Lung RSV Titers. Viral titers from total lung and nose homogenates weremeasured on days 4 and 10 post-intranasal challenge with approximately5.0 Log₁₀ of RSV/A/Long. On day 4 post-infection viral load in RSVinfected normal and immunosuppressed cotton rats treated with saline(groups D and A, respectively) was comparable between the groups forboth lungs and noses. Therapeutic treatment with IVIG of either normal(groups E and F) or immunosuppressed animals (groups B and C) resultedin a statistically significant (p<0.05) reduction of lung and nose viraltiter. On day 10 post-infection, no RSV was detected in the lungs ofsaline-treated normal infected animals (group D), and virus was barelydetectable in the nose of two out of the five group D animals. Incontrast, over 6 Log₁₀ PFU/gram was recovered from the lungs and nose ofsaline-treated immunosuppressed animals infected with RSV (group A).IVIG treatment caused in a statistically-significant reduction of viralload in both the lungs and the nose of infected animals (groups B andC). A moderate dose-dependency was seen, with 0.5 Log₁₀ PFU/gram greaterreduction seen in the lung samples collected from the group of animalstreated with the higher dose of IVIG (group B compared to group C) (SeeFIG. 8).

RSV detection by qPCR. RSV gene expression (NS1 mRNA) was quantified byqPCR assay in the lung, liver, and kidney samples collected from groupA, B, C, and D animals on day 10 p.i. The level of RSV transcript waslowest in group D and highest in group A samples. Treatment ofimmunosuppressed animals with IVIG resulted in statistically-significantreduction in the lung RSV NS1 mRNA level (groups B and C compared togroup A) (FIG. 9). NS1 transcript was also reduced by IVIG treatment inthe liver and kidney of immunosuppressed RSV-infected animals.

Immunosuppression in cyclophosphamide-treated groups A, B, C, and G wasverified by significant decrease in whole blood cell and lymphocytecounts compared to normal cotton rats (groups D, E, and F).Immunosuppressed animals treated with IVIG of the invention, groups Band C, showed significant reduction in lung and nose viral load at day 4p.i. and day 10 p.i. compared to RSV-infected immunosuppressed animalstreated with saline (group A). This reduction was accompanied byreduction in lung histopathology and a decrease in the detection ofviral RNA in lung, liver, and kidney samples of group B and C animals onday 10 p.i. Treatment of cotton rats with IVIG of the invention resultedin significant reduction of RSV load in the lungs and nose ofIVIG-treated animals (groups E and F) compared to saline-treated (groupD) animals on day 4 p.i.

Example 4 IVIG Prophylactic Potential

Immunotherapeutic compositions of the invention were tested in a cottonrat model of human immunodeficiency. Therapeutic as well as prophylacticpotential was assessed. For analysis of prophylactic potential,immunoglobulin prepared as described in Example 2 above was usedprophylactically in immunosuppressed (cyclophosphamide treated) cottonrats challenged with RSV/A/Long.

Animals: Thirty (30) inbred male and female Sigmodon hispidus cottonrats between 6 to 8 weeks of age (Source: Sigmovir Biosystems, Inc.,Rockville Md.) were maintained and handled under veterinary supervisionin accordance with the National Institutes of Health guidelines andSigmovir Institutional Animal Care Utilization Committee's approvedanimal study protocol (IACUC Protocol #2). Cotton rats were housed inclear polycarbonate cages individually and provided with standard rodentchow (Harlan #7004) and tap water ad lib.

Challenge Virus: The prototype Long strain of RSV (ATCC, Manassas, Va.)was propagated in HEp-2 cells after serial plaque-purification to reducedefective-interfering particles. A pool of virus designated ashRSV/A/Long Lot #041513 containing approximately 5.0×10₇ pfu/mL insucrose stabilizing media was used for in vivo experiments. This stockof virus is stored under −80° C. conditions and has been characterizedin vivo using the cotton rat model and validated for upper and lowerrespiratory tract replication.

Methods.

Identification of animals was performed using ear tags. Bleeding wasperformed using retro-orbital sinus bleed. Collection in EDTA tubes forwhole blood analysis (day −3), collection of serum (prebleed, day 4p.i., day 10 p.i.). Route of infection was intranasal (i.n.)inoculation. Route of IVIG treatment was intra-peritoneal injection(i.p.). Euthanasia was performed using CO2 asphyxiation. Lung tissue,liver tissue, and kidney tissue were harvested post euthanasia for RSVplaque assays, quantitative PCR (qPCR) and histopathology.

Table 5 provides experimental study design for these experiments.

TABLE 5 Valente of 10% IgG stock solution # of administered i.p. GroupAnimals Treatment Route Dose per 100 g weight: Sac CY-treated: A 10Normal Saline i.p. 1.5 ml 1.5 ml d 4, d 10 B 10 Anti-RSV IgG 1 i.p. 1500mg/kg 1.5 ml d 4, d 10 C 10 Anti-RSV IgG 1 i.p. 750 mg/kg 0.75 ml  d 4,d 10

FIG. 5 shows a schematic of a cotton rat model utilized to study theprophylactic potential of an immunoglobulin prepared as described inExample 2.

Whole Blood Assay. Automated whole blood analysis was carried out onblood samples collected in EDTA-containing tubes. Total number of whiteblood cells and lymphocytes was analyzed and presented in comparison tohistorical blood values of normal, unmanipulated, age-matched cottonrats.

Serum total IgG ELISA. Ninety-six well plates were coated with 1:1,000rabbit anti-cotton rat IgG in coating solution (KPL 50-84-00). Wellswere subsequently blocked (KPL 50-61-00) and serum samples diluted1:50,000 were loaded. Bound cotton rat IgG was detected with chickenanti-cotton rat IgG (1:15,000; Immunology Consultants LaboratoryCCOT-25A), followed by HRP-labeled goat anti-chicken IgG (1:10,000;KPL). The amount of IgG in each serum sample was quantified with respectto the standard curve constructed of 10-fold dilution of normal cottonrat serum, with 1:100 dilution of serum assumed to have 10,000 Units ofIgG.

RSV plaque assay. Lung homogenates were clarified by centrifugation anddiluted 1:10 and 1:100 in EMEM. Confluent HEp-2 monolayers in 24-wellplates were infected in duplicates with 50 μl of sample per wellstarting with undiluted (neat) samples followed by diluted homogenates.After one hour incubation at 37° C. in a 5% CO₂ incubator, wells wereoverlayed with 0.75% methylcellulose medium and plates restored into the37° C. incubator. After 4 days of incubation the overlay was removed andthe cells were fixed with 0.1% crystal violet stain for one hour, thenrinsed and air-dried. Plaques were counted and viral titers wereexpressed as plaque forming units per gram of tissue. Viral titer for agroup was calculated as the geometric mean+standard error for allanimals in that group at a given time. Student-t test was applied todetermine significance of change in viral replication betweenvehicle-treated and test groups, with p<0.05 indicating astatistically-significant difference.

Real-time PCR. Total RNA was extracted from homogenized lung, kidney orliver tissue using the RNeasy purification kit (QIAGEN). One μg of totalRNA was used to prepare cDNA using QuantiTect Reverse Transcription Kit(Qiagen). For the real-time PCR reactions the QuantiFast SYBR Green PCRKit (Qiagen) was used in a final volume of 25 with final primerconcentrations of 0.5 μM. Reactions were set up in 96-well trays.Amplifications were performed on a Bio-Rad iCycler for 1 cycle of 95° C.for 3 min, followed by 40 cycles of 95° C. for 10 sec, 60° C. for 10sec, and 72° C. for 15 sec. The baseline cycles and cycle threshold (Ct)were calculated by the iQ5 software in the PCR Base Line SubtractedCurve Fit mode. Relative quantification of DNA was applied to allsamples. The standard curves were developed using serially-diluted cDNAsample most enriched in the transcript of interest (e.g., lungs from day4 post-primary RSV infection). The Ct values were plotted against log₁₀cDNA dilution factor. These curves were used to convert the Ct valuesobtained for different samples to relative expression units. Theserelative expression units were then normalized to the level of ®-actinmRNA (“housekeeping gene”) expressed in the corresponding sample. Foranimal studies, mRNA levels were expressed as the geometric mean±SEM forall animals in a group at a given time.

Results.

Whole Blood Assay. Total White Blood Cell counts and total Lymphocytecounts were reduced in all cyclophosphamide treated animals compared tonormal, unmanipulated cotton rats (historical WBC and Lymphocyte countvalues for 6-8 week-old S. hispidus were used for comparison). Thisdifference was seen in samples collected 18 days after the beginning ofcyclophosphamide treatment (Day −3 with respect to RSV challenge, FIG.10A), as well as in samples collected from animals sacrificed on Day 4and Day 10 post-RSV-challenge (FIG. 10B).

Serum Total IgG ELISA. The level of total IgG in the serum ofcyclophosphamide-treated cotton rats was reduced compared to serum ofcontrol animals (FIG. 11). The reduction was seen for serum collected onboth days analyzed: 3 days prior to and 10 days after infection, whichcorresponds to days 18 and 31 after the beginning of cyclophosphamidetreatments.

Lung RSV Titers. Viral titers from total lung homogenates were measuredon days 4 and 10 post-intranasal challenge with approximately 5.0 Log₁₀of RSV/A/Long. On day 4 post-infection 4.98 Log 10 PFU/g RSV wasdetected in the lungs of animals from Group A (FIG. 12). No virus wasrecovered from the lungs of animals in groups B and C. On day 10post-infection, 6.14 Log 10 PFU/g RSV was detected in the lungs ofanimals from Group A. No virus was recovered from the lungs of animalsin Group B, and in 3 out of 5 animals in group C.

Kidney and Liver RSV Titers. RSV infectious viral particles werequantified by plaque assay in the kidney and liver of one animal fromGroup A (animal #96998) and one animal from Group B (animal #97009). Novirus was detected in either organ of either animal. RSV detection byqPCR. RSV gene expression (NS1 mRNA) was quantified by qPCR assay in thelung, kidney and liver of one animal from Group A (animal #96998) andone animal from Group B (animal #97009). NS1 mRNA expression wasdetected in all three organs collected from the animal in Group A, andin the lung and liver of animal in Group B (FIG. 13). For all threeorgans analyzed, RSV gene expression was higher in the animal in Group Aanimal compared to the animal in Group B.

Histopathology. Histopathology was analyzed in the lung, kidney andliver samples of one animal from Group A (animal #96998) and one animalfrom Group B (animal #97009). No significant differences were notedbetween the Group A and B kidney samples and between Group A and B liversamples analyzed. Pulmonary pathology in the Group A animal was strongerthan in the Group B animal and was characterized by thickening anddegeneration of bronchiolar epithelium, interstitial pneumonia, and mildalveolitis.

Immunosuppresion was verified by significant decrease in whole bloodcell and lymphocyte counts, and reduction in serum total IgG.Immunosuppressed animals treated with IVIG showed undetectable lungviral replication at day 4 p.i., and almost complete reduction in theprolonged viral replication caused by immunosuppression measured on day10 (only two out of 5 animals in a group showed minimal viralreplication). This reduction was accompanied by reduction in lunghistopathology and decrease in the detection of viral RNA in lung,kidney, and liver samples of selected immunosuppressed animals.

Various modification, recombination, and variation of the describedfeatures and embodiments will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughspecific embodiments have been described, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes andembodiments that are obvious to those skilled in the relevant fields areintended to be within the scope of the following claims. Allpublications and patents mentioned in the present application and/orlisted below are herein incorporated by reference in their entireties.

1-39. (canceled)
 40. A method of treating a respiratory infection in asubject comprising administering to the subject a therapeuticallyeffective amount of an immunotherapeutic composition comprising: A)immune globulin prepared from a pooled plasma composition comprisingplasma samples from 500 or more human plasma donors, wherein the pooledplasma composition has a final respiratory syncytial virus (RSV)neutralization titer of at least 1800, and an antibody titer forparainfluenza virus 1, parainfluenza virus 2, influenza A virus, and/orinfluenza B virus that is at least 1.3 times greater than the antibodytiter in a control sample, wherein the control sample is a mixture ofplasma samples obtained from 500 or more random human plasma donors, andwherein less than 50% of the donor plasma samples used for pooling arefrom donors with a RSV neutralization titer of at least 1800; and B) apharmaceutically acceptable carrier.
 41. The method of claim 40, whereinthe respiratory infection is an upper respiratory tract infection. 42.The method of claim 40, wherein the respiratory infection is a lowerrespiratory tract infection.
 43. The method of claim 40, wherein therespiratory infection is caused by a pathogen selected from the groupconsisting of respiratory syncytial virus (RSV), parainfluenza virus,coronavirus, influenza virus, and metapneumovirus.
 44. The method ofclaim 40, wherein the treating reduces viral load in the subject. 45.The method of claim 44, wherein the treating reduces the viral load inthe lungs of the subject.
 46. The method of claim 40, wherein thetreating reduces the level of pathogenic viral RNA in an organ of thesubject.
 47. The method of claim 46, wherein the organ is selected fromthe lungs, liver and kidneys.
 48. The method of claim 40, wherein thesubject is selected from the group consisting of an end stage renaldisease (ESRD) patient, a patient on immunosuppressive therapy, an AIDSpatient, a diabetic patient, a neonate, a transplant patient, a patientwith malfunctioning immune system, an elderly person, a patient withautoimmune disease, a burn patient, a cancer patient, and a patient inan acute care setting.
 49. The method of claim 40, wherein theimmunotherapeutic composition comprises a recombinant monoclonalantibody specific for a respiratory pathogen, a recombinant polyclonalantibody specific for a respiratory pathogen, or combination thereof.50. The method of claim 49, wherein the respiratory pathogen is selectedfrom the group consisting of respiratory syncytial virus, influenza Avirus, influenza B virus, influenza C virus, parainfluenza virus type 1,parainfluenza virus type 2, rhinovirus, metapneumovirus, coronavirus, S.pneumonia, H. influenza, L. pneumophila, group A Streptococcus,Streptococcus mutans, B. gingivalis, S. pyogenes (group A), S.pneumoniae, K pneumoniae, P. aeruginosa, S. aureus, and M. pneumoniae.51. The method of claim 40, wherein the immunotherapeutic compositioncomprises an anti-toxin agent.
 52. The method of claim 51, wherein theanti-toxin agent is a mono-specific, bi-specific or multi-specificantibody with specificity toward a bacterial or fungal toxin.
 53. Themethod of claim 52, wherein the bacterial or fungal toxin is selectedfrom the group consisting of Botulinum neurotoxin, Tetanus toxin, E.coli toxin, Clostridium difficile toxin, Vibrio RTX toxin,Staphylococcal toxins, Cyanobacteria toxin, and mycotoxins.
 54. Themethod of claim 40, wherein the immunotherapeutic composition comprisesneutralizing antibodies specific for one or more of Corynebacteriumdiphtheria, measles virus, and polio virus.
 55. The method of claim 40,wherein the immunotherapeutic composition comprises an agent selectedfrom the group consisting of an anti-inflammatory agent, an anti-canceragent, an anti-microbial agent, an antiviral agent, an immunotherapeuticprotein, an immunotherapeutic small molecule, an antihistamine, acytokine, and a chemokine.
 56. The method of claim 55, wherein theanti-inflammatory agent is selected from the group consisting of arecombinant antibody, an antibody fragment, a monoclonal antibody, ananti-inflammatory protein and an anti-inflammatory small molecule.